CN100369925C - Hyaluronic acid oligosaccharide fractions and drugs containing the same - Google Patents

Hyaluronic acid oligosaccharide fractions and drugs containing the same Download PDF

Info

Publication number
CN100369925C
CN100369925C CNB018124321A CN01812432A CN100369925C CN 100369925 C CN100369925 C CN 100369925C CN B018124321 A CNB018124321 A CN B018124321A CN 01812432 A CN01812432 A CN 01812432A CN 100369925 C CN100369925 C CN 100369925C
Authority
CN
China
Prior art keywords
oligose
fraction
cell
sugar
hyaluronic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CNB018124321A
Other languages
Chinese (zh)
Other versions
CN1440418A (en
Inventor
浅利晃
栗原仁
柴田知己
宫崎由佳
山之口裕子
多和田明
政孝浩
松崎祐二
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Seikagaku Corp
Original Assignee
Seikagaku Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Seikagaku Corp filed Critical Seikagaku Corp
Publication of CN1440418A publication Critical patent/CN1440418A/en
Application granted granted Critical
Publication of CN100369925C publication Critical patent/CN100369925C/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H7/00—Compounds containing non-saccharide radicals linked to saccharide radicals by a carbon-to-carbon bond
    • C07H7/02—Acyclic radicals
    • C07H7/033—Uronic acids

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Biochemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Dermatology (AREA)
  • Materials Engineering (AREA)
  • Polymers & Plastics (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Saccharide Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention provides hyaluronic acid oligosaccharides having a size selected from sizes of 4 to 60 saccharides, fractions containing the hyaluronic acid oligosaccharides and having particular physicochemical properties, and drugs containing the same. The hyaluronic acid oligosaccharides of the present invention are extremely useful, because they exert superior pharmaceutical effects as active ingredients of a heat shock protein expression promoter, cell death inhibitor, cell injury inhibitor and cell and tissue protecting agent (e.g., organ preservation agent, antiulcer agent, antihepatopathic agent, IL-10 production promoter or IL-8 production inhibitor) and exhibit high safety.

Description

Hyaluronic acid oligosaccharide fraction and contain the medicine of this material
Technical field
The present invention relates to hyaluronic acid oligosaccharide (hereinafter referred to as " HA oligose ") and new fraction thereof.
And the medicine that to the present invention also relates to this HA oligose be effective constituent is (especially for heat shock protein expression facilitator, cell death inhibitor, cell obstacle inhibitor; or cell, organization protection's agent (particularly organ preservatives, ulcer treatment agent, and liver obstacle treatment agent, IL-10 produces promotor, or IL-8 generation inhibitor).
Background technology
Hyaluronic acid (hereinafter referred to as " HA "), it is known that its activity or function change with its molecular size.For example, molecular weight is [High molecular weight hyaluronic acid inhibitsadvanced glycation endproduct-induced NF-kappaB activation andcytokine expression.Neumann A such as passivation with NF-κ b of 1,200,000 HA, angiogenesis restraining effect, Schinzel R, Palm D, Riederer P, MunchG.FEBS Lett 1999 Jun 25; 453 (3): 283-7; Feinberg RN, Beebe DC.Hyaluronate in vasculogenesis.Science 220:1177-1179,1983.], molecular weight is that the HA below 500,000 it is reported to have active in contrast to this [Noble PW, McKee CM, Cowman M, Shin HS.Hyaluronan fragments activate an NF-kappaB/I-kappaB alpha autoregulatory loop in murine macrophages.J Exp Med183:2373-2378,1996.; West DC, Shaw DM.Tumour hyaluronan inrelation to angiogenesis and metastasis.In:Laurent TC, ed.The chemistry, biology and medical applications of hyaluronan and its derivatives.London:portland press, 1998:227.].
These true fully expression HA oligose may have various activity, or might find the activity that it is special according to the size of HA oligose.Thereby during the different HA oligose of combined size, the effect that may give play to addition or multiply each other in contrast, for the activity of the HA oligose of a certain size, will consider that also other big or small HA oligose produce the effect of antagonists.
At this moment, at first towards the activity research of the drug development of HA oligose in the stage, use the HA oligose blended level timesharing of all size, HA oligose that not only can not determine which kind of size is an active main body, and have the activity of the HA oligose of a certain size to be offset by the HA oligose of different sizes, might miss the important physiologically active or the function that are hidden in the HA oligose.
In addition, when the HA oligose of a certain size is used as medicine, be necessary to get rid of other big or small oligose that influences this HA oligose performance function as far as possible, and, the highly purified fraction of inessential material in fact do not contained as the medicine needs.
As mentioned above, in new medicine development and providing, need constitute and not contain in fact by the HA oligose of single size in fact the high purity HA oligose fraction of other big or small HA oligose or other impurity.
On the other hand, heat shock protein (hereinafter referred to as Hsp) is also referred to as stress protein matter, is the protein of a kind of defence because of the obstacle that various stress response produced, known multiple family.The Hsp70 family heat shock protein of one of them, can suppress the factor because of environmental stress generations such as thermal shocking, hydrogen peroxide, heavy metal, amino acid analogue, scarce sugar, or heating, inflammation, ischemic, virus infection, metabolic trouble, megalocardia disease, oxidation stress, cell tissue obstacle, oncogene or the carcinogenic substance equal stress factor and the proteinic structural changes that produces, with generation of abnormal protein etc., by making proteinic functional regeneration etc., can prevent cell obstacle or necrocytosis simultaneously.
About these contents, in the flat 9-227386 communique of Japanese patent application, put down in writing the stress protein matter expression facilitator as effective constituent with HA.In this communique, put down in writing HA (page 3) as preferred 2～20 sugar of its activeconstituents, the following HA of hint 10 sugar participates in the enhancing of stress protein matter expression or induces (the 6th page).And then also put down in writing and represented that the unsaturated disaccharides of HA strengthens the expression of stress protein matter, suppresses the experiment (embodiment 2) of necrocytosis.
But the various oligose for the present invention relates to do not have concrete disclosing, and for following in HA4 sugar the extremely significant stress protein matter of special discovery express and strengthen, also without any open or hint.
And, from the function of aforementioned stress protein matter, can infer that stress protein matter participates in the protection of cell, tissue.
About these contents, in the flat 11-246301 communique of Japanese patent application, put down in writing the organ preservation solutions of the transplanting usefulness that contains the salt that allows on HA and/or the physiology.In this communique, the molecular-weight average of having put down in writing HA is preferred more than 100,000, but does not illustrate or hint for the excellent results of HA oligose and performance thereof.
Summary of the invention
The object of the present invention is to provide to have 4 sugar～the big or small HA oligose of 60 sugar, particularly, the HA oligose by single size constitutes, does not contain other the big or small HA oligose or the fraction of other impurity in fact.
Another object of the present invention is to provide with the medicine of such HA oligose as effective constituent, particularly, provides Hsp expression facilitator, cell death inhibitor and the cell obstacle inhibitor of better effects if.And then, the object of the present invention is to provide good cell, organization protection's agent, utilize organ preservatives, ulcer treatment agent, hepatic diseases treatment agent, the IL-10 of this material to produce promotor and IL-8 generation inhibitor.
The present inventor for the result who solves above-mentioned problem and concentrate on studies is, the HA oligose of 4 sugar～60 sugar will be selected from, and the mixture of these oligose, separate classification by special methods, obtained constituting and not containing by the oligose of target sizes in fact the new fraction of other big or small oligose and other impurity.
That is to say, the invention provides and contain oligose of the present invention, and feature is the fraction (hereinafter referred to as " fraction of the present invention ") with physico-chemical property shown in following (1)～(6).
(1) with this fraction, utilize gel filtration chromatography, with following (a) and (b) described detection method analyze, its result is no matter show single in fact peak with any method, and as described in the area at this peak reaches (b) as following (a).
When (a) utilizing the 210nm absorption detecting, with respect to the peak area sum of whole HA oligose in the fraction, the relative area at aforementioned single in fact peak is more than 85%.
When (b) utilizing differential refractometer to detect, with respect to the peak area sum of whole HA oligose in the fraction, the relative area at aforementioned single in fact peak is more than 98%.
(2) with this fraction, utilize anion-exchange chromatography, when analyzing, show single in fact peak, and with respect to the peak area sum of whole HA oligose in the fraction, the relative area at aforementioned single in fact peak is more than 90% with the 210nm absorption detecting.
(3) this fraction is carried out when analyzing, show single bands of a spectrum after the fluorescent mark by electrophoresis, and, do not detect the bands of a spectrum of other big or small HA oligose.
(4) will constitute the single isotopic molecule amount of HA oligose of this fraction or the theoretical value of molecular-weight average and be set at 1 o'clock, the measured value of the mass spectroscopy of this fraction is 0.997～1.003 (relative value).
(5) constitute the theoretical value (weight %) of the ratio of content separately of carbon (C), hydrogen (H) and nitrogen (N) in the HA oligose of this fraction, the measured value (weight %) of each element that obtains with ultimate analysis by this fraction poor is all in ± 1 (weight %) scope.
(6) do not contain protein, DNA and intracellular toxin in fact.
And then, the physico-chemical property shown in (7) below fraction of the present invention preferably has.
(7) this fraction 1H-NMR reaches 13The C-NMR measurement result does not produce contradiction with the structure of the HA oligose shown in the following formula (1).
Formula (1)
Figure C0181243200081
(in the formula, n represents 1～29 integer, and M represents proton or monovalent cation, and Ac represents ethanoyl)
The size of the HA oligose that contains in the fraction of the present invention is preferably selected from 4 sugar～20 sugar, is more preferably to be selected from 4 sugar～16 sugar, further is preferably selected from 4 sugar～14 sugar, preferred especially 4 sugar.
In addition, the present inventor has studied the possibility of oligose of the present invention as medicine, its result, the HA oligose of finding prescribed level has extremely significant Hsp and expresses enhancement, necrocytosis restraining effect and cell obstacle restraining effect, the Hsp expression facilitator, cell death inhibitor and the cell obstacle inhibitor that utilize this discovery to provide to solve above-mentioned problem.
And the present inventor finds the superior cell of oligose performance of the present invention, organization protection's effect; thereby cell, organization protection's agent are provided, and then provide the organ preservatives, ulcer treatment agent, hepatopathy treatment agent, the IL-10 that utilize this material to produce promotor and IL-8 generation inhibitor.
That is to say, the invention provides the medicine (medical composition is hereinafter referred to as " medicine of the present invention ") of oligose of the present invention as effective constituent.This medicine is preferably Hsp expression facilitator, cell death inhibitor, cell obstacle inhibitor, or cell, organization protection's agent.
And cell, organization protection's agent, preferably produce promotor or IL-8 and produce inhibitor and use as organ preservatives, ulcer treatment agent, hepatopathy treatment agent, IL-10.
Medicine of the present invention and these medicaments, the HA oligose that preferably will be selected from 4 sugar～20 sugar sizes is as effective constituent, the HA oligose that more preferably will be selected from 4 sugar～16 sugar sizes is as effective constituent, further be more preferably and will be selected from the big or small HA oligose of 4 sugar～14 sugar as effective constituent, preferred especially HA4 sugar is as effective constituent.
As mentioned above, existed about the physiologically active of HA oligose, the report of function in the past.But be to use from molecular size, molecular structure, the purity (degree of sneaking into of other molecular sizes, the degree of sneaking into of other compositions such as protein, DNA, endotoxin content) example of the HA oligose that guarantees of multiple angles such as, so far also do not occur, that is to say, in the report of the physiologically active of in the past HA oligose, function, negate the influence of tramp material.To this in the present invention, obtained not containing the HA oligose of other compositions such as protein, DNA in fact, and then obtained not containing in fact the fraction of HA oligose of the specific size of other molecular sizes, the clear and definite physiologically active of these materials, function.
Below, describe the present invention in detail.
＜1〉oligose of the present invention
Oligose of the present invention is to have the HA oligose that is selected from 4 sugar～60 sugar sizes.
So-called " HA oligose " is by forming the identical oligose that constitutes of forming with the disaccharides that constitutes of HA.Specifically, be alternately with glycosidic linkage bonded oligose as the glucuronic acid (GlcA) of the formation monose of HA and N-acetyl glucosamine (GlcNAc).
That is, structure shown in the aforementioned formula (1) represents that not only non-reduced end is the oligose of glucuronic acid, comprises that also non-reduced end is the HA oligose of N-acetyl glucosamine.
Being in the sugar on the non-reduced end, can be saturated sugar (carbon-carbon bond in the monose does not comprise two keys), also can be unsaturated sugar (carbon-carbon bond in the monose comprises two keys).In the following description, GlcA represents saturated glucuronic acid, and Δ GlcA represents unsaturated glucuronic acid.
The sugar that wherein especially preferably is on the non-reduced end is saturated sugar, specifically, and the HA oligose shown in the preferred following formula (2).
GlcA(-GlcNAc-GlcA) n-GlcNAc (2)
(in the formula, GlcA represents glucuronic acid residue, and GlcNAc represents N-acetyl glucosamine residue ,-expression glycosidic linkage, n represents 1～29 integer)
Preferred β 1 → 3 key of the glycosidic linkage of GlcA-GlcNAc in following formula (2), preferred β 1 → 4 key of the glycosidic linkage of GlcNAc-GlcA.
Wherein, the HA oligose shown in the preferred especially following formula (1).
Formula (1)
Figure C0181243200101
(in the formula, n represents 1～29 integer, and M represents proton or monovalent cation, and Ac represents ethanoyl)
As described later shown in the embodiment, when being in sugar on the non-reduced end and being unsaturated sugar, also show significant physiologically active, these also are the preferred embodiment of the present invention.As a concrete example, can exemplify the HA oligose shown in the following formula.
Figure C0181243200102
(in the formula, Ac represents ethanoyl)
The HA oligose that contains unsaturated sugar in the non-reduced end shown in the following formula is equivalent to the Δ HA4 that uses among the aftermentioned embodiment.
And oligose of the present invention can be the form of salt, also can be ionized state.As salt, for example salt of mineral alkalis such as an alkali metal salt (sodium salt, lithium salts, sylvite etc.), alkaline earth salt, ammonium salt is arranged, or the salt of organic bases such as diethanolamine salt, cyclohexylamine salt, amino acid salts, semi-lactosi amine salt, glucosamine salt.Preferred as alkali salt wherein, special particular certain cancers.
There is no particular limitation in the source of oligose of the present invention.For example, the skin of cockscomb, umbilical cord, pigskin, ox-hide, fish and other animals, Aorta etc., or separate from the microorganism that produces HA by disaggregating treatment (for example enzyme decomposition, decomposition, heat treated, ultrasonication etc.). the material of the step preparation of the HA of purifying.And, also can be material by the step preparation of synthetic (for example chemosynthesis or enzymic synthesis).
As the enzyme decomposition method, can exemplify decomposition Unidasa (from testis), Unidasa (from streptomycete), Unidasa SD, chondroitinase ACI, chondroitinase ACIII, chondroitinase abc, the enzyme of interior glucuronidase HA such as (from leech) acts on the method for HA (with reference to neonatology experiment lecture " saccharic II-protein-polysaccharide and mucopolysaccharide " 244-288 page or leaf, distribution in 1991, the same people of Tokyo chemistry).In order to obtain the oligose shown in the following formula (1),, preferably use lytic enzyme as the enzyme of decomposing H A.
As chemical decomposition method, can exemplify caustic leaching process or dimethyl sulfoxide method (DMSO method).Caustic leaching process, specifically, for example by add alkali such as 1N sodium hydroxide in the solution of HA, heating through a few hours makes it degraded, adds sour neutral mode such as hydrochloric acid afterwards and carries out.As the DMSO method, can exemplify the method (Carbohyd.Res., 141,99-110 page or leaf, 1985) of Nagasawa etc.And, also can be hydrolyzed by hydrochloric acid or sulfuric acid.
As the ultrasonication method, can exemplify at Biochem., 33, the method for record in the 6503-6507 page or leaf (1994) etc.
As synthesis preparation method, can exemplify Glycoconjugate J, 453-439 page or leaf (1993), the method for records such as international open WO93/20827.
Zhi Bei oligose of the present invention can be purified to required purity like this.For example the following describes, the oligose of the present invention that can be purified in fact by single size constitutes, and does not contain the degree of other big or small HA oligose and other impurity.
＜2〉fraction of the present invention
Fraction of the present invention for containing the fraction of oligose of the present invention, is the material with following physico-chemical property.
By aforementioned＜1〉record method can obtain the oligose of the present invention (fraction that contains oligose of the present invention.Different with " fraction of the present invention "), but in the fraction that obtains, be mixed with the HA oligose of multiple size.On the other hand, fraction of the present invention is to be made of the HA oligose of single size in fact, and does not contain the fraction of other big or small HA oligose and other impurity.Fraction of the present invention can prepare by the following method.
Will be by aforementioned＜1〉fraction (being mixed with the fraction of all size oligose) that the method for record obtains, be loaded on the post of strongly basic anionite.As strongly basic anionite, can exemplify the anionite that contains trimethylammonio, trimethyl ammonium methyl, beta-hydroxyethyl diformazan ammonium, 2-hydroxy propyl amido, diethyl-(2-hydroxypropyl) aminoethyl, dimethyl ethanol ammonium etc., but preferably contain the anionite of trimethyl ammonium methyl.
The size of post can be according to suitably settings such as applied amounts, and the diameter of preferred post is 1～5cm when preparing on a small scale, and the length of post is 50～150cm.And preferably combination is used this post more than 2.
When being loaded into the fraction that is mixed with multiple big or small HA oligose on such post, HA oligose that contains in the fraction and the strongly basic anionite ionic bond in the post.
Bonded HA oligose can wash-out according to the concentration gradient of salt.There is no particular limitation to be used for the salt of wash-out, can use NaCl, KCl, LiCl etc., preferably uses NaCl.
The concentration gradient of salt in addition, preferably salt concentration rises to about 0.5M from about 0～0.1M.And the concentration gradient of preferably salt is straight line, preferably with the increase salt concn of 0.1M/40～50 hour, is more preferably the speed rising salt concn with 0.1M/45～50 hour.
Preferred 80～the 100ml/ of flow velocity hour in addition, be more preferably 85～95ml/ hour.
By selecting such chromatographic condition, the HA oligose of ionic bond on strongly basic anionite is from big slight beginning wash-out successively.The bonding force that can know HA oligose and strong basicity ion-exchanger thus roughly with being in proportion of HA oligose, its result, because of the load of salt concn gradient, oligose (the big slight oligose) beginning a little less than the bonding force is by wash-out successively.Fraction of the present invention is by utilizing the combining of such strongly basic anionite and HA oligose, wash-out characteristic to provide first.According to this method, utilize high separation energy, and by 1 column operation, HA oligose that can the simple separation all size, the HA oligose that can effectively prepare in fact by single size constitutes, and does not contain the highly purified HA oligose fraction (fraction of the present invention) of other big or small HA oligose or other impurity.
The HA oligose that contains in the fraction of the present invention is preferably selected from 4 sugar～20 sugar sizes, is more preferably and is selected from 4 sugar～16 sugar sizes, further is preferably selected from 4 sugar～14 sugar sizes, preferred especially 4 sugar.
The fraction of the present invention that obtains like this can be attached in the above-mentioned chromatographic step once more.And, also can be attached to concentrated, desalination, and during other handle.
The preservation of fraction of the present invention, circulation form do not have special qualification, can be in solution state, frozen state, the lyophilised state any one.
And the HA oligose in the fraction of the present invention can be the form of salt, also can be ionized state.As salt, for example salt of mineral alkalis such as an alkali metal salt (sodium salt, lithium salts, sylvite etc.), alkaline earth salt, ammonium salt is arranged, or the salt of organic bases such as diethanolamine salt, cyclohexylamine salt, amino acid salts, semi-lactosi amine salt, glucosamine salt.Preferred as alkali salt especially wherein, special particular certain cancers.When fraction of the present invention is used for aftermentioned medicine of the present invention, can uses in these salt and medically allow the salt that uses.Also special at this moment particular certain cancers.
The fraction of the present invention that obtains like this, all physico-chemical properties shown in (1)～(6) below it is characterized in that satisfying.In addition, the numerical value shown in the following physico-chemical property etc., according to test or experiment condition, environment, use equipment, trier's proficiency, and other factors etc. may have some variations.Thereby this numerical value does not have strictness to be defined on the data, should consider according to measurement results such as experiment conditions there are differences (the numerical value difference in those skilled in the art's general knowledge scope; Those skilled in the art can regard as the numerical value difference of same fraction degree).
(1) utilize gel filtration chromatography, with following (a) and (b) described detection method analyze, its result, no matter all show single in fact peak, and as described in the area at this peak reaches (b) as following (a) with any method,
During (a) based on the absorption detecting of 210nm, with respect to whole peak area sum of HA oligose in the fraction, the relative area at aforementioned single in fact peak is more than 85%.This relative area, preferred more than 90%, be more preferably more than 95%.
When (b) utilizing differential refractometer (following also slightly be called [RI]) to detect, the peak area sum of whole HA oligose in the relative fraction, the relative area at aforementioned single in fact peak is more than 98%.
This physico-chemical property represents that this fraction is made of the HA oligosaccharides molecule of single in fact size.And expression does not contain other impurity in fact.
In addition, so-called " single in fact " is not the implication of " single fully " among the present invention, but the implication of " those skilled in the art can regard as single ".For example, general, be difficult to reach in the bigger oligose of size and do not contain other big or small oligose fully, this can be described as the general knowledge in present technique field.Therefore, in the stratographic analysis of the bigger oligose fraction of size or mass spectroscopy etc., except the main peak that the bigger oligose of size produces, even the small peak that exists other big or small oligose to produce, when considering the oligose size, also assert it is single in fact peak.Thereby, can be called " single in fact peak " under such situation.
(2) with this fraction, utilize anion-exchange chromatography, when analyzing, express single in fact peak with the 210nm absorption detecting, and, the peak area sum of whole HA oligose in the relative fraction, the relative area at aforementioned single in fact peak is more than 90%.This relative area is preferably more than 95%.
This physico-chemical property represents that this fraction is made of the HA oligosaccharides molecule with single in fact electric charge, and promptly the HA oligose by homogeneous constitutes.And expression does not contain other impurity in fact.
In addition, for example shown in the formula (1), have the carboxyl of rule in the HA oligosaccharides molecule, this group is ionized in solution-COO -Ion.Thereby, we can say contain in a certain size the HA oligosaccharides molecule-COO -Number of ions is certain.Therefore, the electric charge of the HA oligose of formation fraction is single in fact, illustrates that also the HA oligose by single in fact size constitutes.
(3) this fraction is carried out when analyzing, express single bands of a spectrum after the fluorescent mark by electrophoresis, and, do not detect the bands of a spectrum of other big or small HA oligose.
Even this presentation of results is analyzed by the method (electrophoretic method) beyond the chromatography, also can clear and definite this fraction constitute by the HA oligosaccharides molecule of single in fact size, also support the result of chromatography analysis.And expression does not contain other impurity in fact.
(4) the single isotopic molecule amount or the molecular-weight average theoretical value that will constitute the HA oligose of this fraction is set at 1 o'clock, and the measured value of the mass spectroscopy of this fraction is 0.997～1.003 (relative value).This relative value, preferred 0.998～1.002 scope is more preferably 0.999～1.001 scope.
This presentation of results constitutes the HA oligose of fraction, and its molecular weight (quality) is also single in fact, and hint does not contain other impurity in fact.
(5) constitute the theoretical value (weight %) of the ratio of content separately of carbon (C), hydrogen (H) and nitrogen (N) in the HA oligose of this fraction, the measured value (weight %) of each element that obtains with ultimate analysis by this fraction poor is all in ± 1 (weight %) scope.
In addition, when the HA oligose is sodium salt, is more preferably for sodium (Na) and is similarly ± 1 (weight %).
This presentation of results constitutes the HA oligose of fraction, and it is elementary composition also single in fact, and, do not contain other impurity in fact.
(6) do not contain protein, DNA and intracellular toxin in fact.
Protein content in the fraction of the present invention can utilize protein determination commonly used as well known to those skilled in the art to measure, but preferred labor profit (Lowry) method.(when the detection boundary is following) do not judged not contain protein in fact when detecting protein by this method.
Dna content in the fraction of the present invention can utilize DNA assay method commonly used as well known to those skilled in the art to measure, but preferred threshold value (threshold) method.(when the detection boundary is following) do not judged not contain DNA in fact when detecting DNA by this method.
Endotoxin content in the fraction of the present invention can utilize endotoxin measurement method commonly used as well known to those skilled in the art to measure, but preferably uses the limulus test of limulus amoebocyte lysate composition.(when the detection boundary is following) do not judged not contain intracellular toxin in fact when detecting intracellular toxin by this method.
What is called in this specification sheets " does not contain in fact ", is not the impurity implication of " molecule does not exist yet ", but expression those skilled in the art can assert degree free from foreign meter (degree that can not detect by above-mentioned detection method).Even analytical technology progress in the future and can detect the branch sub-unit, this " does not contain in fact ", and also should put down in writing content with the application serves as that explain on the basis.
And then in the preferred form of fraction of the present invention, the HA oligose of the single in fact size that contains has saturated glucuronic acid on non-reduced end, and this moment, fraction of the present invention further had following (7) described character.
(7) this fraction 1H-NMR reaches 13The C-NMR measurement result does not produce contradiction with the structure of the HA oligose shown in the following formula (1).
Formula (1)
Figure C0181243200171
(in the formula, n represents 1～29 integer, and M represents proton or monovalent cation, and Ac represents ethanoyl)
The HA oligose that this presentation of results constitutes fraction is a HA oligose shown in the following formula, and its molecular structure is also single in fact, and, do not contain other impurity in fact.
＜3〉medicine of the present invention
Medicine of the present invention is for being the medicine of effective constituent with oligose of the present invention.
As the oligose of the present invention of the effective constituent of medicine of the present invention, or its preferred form etc., as aforementioned＜1〉described.
Wherein, as oligose of the present invention, its size is preferably selected from 4 sugar～20 sugar (HA4 sugar～HA20 sugar), is more preferably the n in the formula (2) before using and is the material that is selected from 1～9 integer, and the n before preferred especially the use in the formula (1) is the material that is selected from 1～9 integer.
Be more preferably, use is selected from the oligose of the present invention (HA4 sugar～HA16 sugar) of 4 sugar～16 sugar sizes, be more preferably the n in the formula (2) before using and be the material that is selected from 1～7 integer, the n before preferred especially the use in the formula (1) is the material that is selected from 1～7 integer.
Be more preferably, use the oligose of the present invention (HA4 sugar～HA14 sugar) be selected from 4 sugar～14 sugar sizes, be more preferably the n in the formula (2) before using and be the material that is selected from 1～6 integer, preferred especially before n in the formula (1) be the material that is selected from 1～6 integer.
Wherein as oligose of the present invention, preferably use 4 sugar substances (HA4 sugar), be more preferably the n in the formula (2) before using and be 1 material, the n before preferred especially the use in the formula (1) is 1 material.By using HA4 sugar, can bring into play various good pharmacological actions.
In addition, preceding formula (1) and (2) are illustrated in the oligose of the present invention that contains glucuronic acid on the non-reduced end, but the concrete purposes of medicine can be used the oligose that contains unsaturated glucuronic acid on non-reduced end according to the present invention, and this moment, preferred its size was also same as above.
Be more preferably, use fraction of the present invention as the effective constituent of medicine of the present invention.With the fraction of the present invention and the preferred form thereof of preparation use, as above-mentioned＜2〉record.And the preferred size of the HA oligose that contains in the fraction of the present invention is also with above-mentioned.
The application of the invention fraction can improve the contained ratio of HA oligose in the medicine of the present invention, and can prepare and do not contain the medicine of the present invention of forbidding the material of sneaking into as medicine in fact.
Medicine of the present invention can be undertaken preparationization by known method.During preparation, can not bring negative impact at effective constituent HA oligose for medicine of the present invention, and in the scope that does not influence medicine effect of the present invention, can use employed other pharmaceutical active ingredients in the common medicine, or stablizer commonly used, emulsifying agent, osmotic pressure conditioning agent, pH regulator agent, buffer reagent, isotonic agent, sanitas, pain killer, tinting material, vehicle, tackiness agent, lubricant, disintegrating agent etc.
And then, medicine of the present invention, preferably contain fraction of the present invention as effective constituent, be selected from the big or small HA oligose of 4 sugar～60 sugar as effective constituent but will have, it is also feasible that such HA oligose contains significant quantity at least, in the scope that does not influence medicine effect of the present invention, even contain the HA oligose of other molecular sizes, or exceed the HA of oligose range size, can be not influenced yet.
Medicine of the present invention, the expression that is preferably used as Hsp strengthen that purposes, necrocytosis suppress purposes, the cell obstacle suppresses purposes, or the medicine of cell, organization protection's purposes, can prepare following preparation according to these purposes.
[1] Hsp expression facilitator
The term of so-called in this specification sheets " express and strengthen " has the double meaning that " expression amount increase " reaches " increased activity ".Therefore, the term of so-called " heat shock protein expression facilitator " (Hsp expression facilitator) in this specification sheets has the double meaning that " preparation that increases the expression amount of heat shock protein (Hsp) " reaches " strengthening the active preparation of Hsp ".
(1) medication of Hsp expression facilitator, formulation etc.
When medicine of the present invention is used as the Hsp expression facilitator, especially preferably use 4 sugar substances in the HA oligose.Like this, can bring into play extremely good Hsp and express enhancement.
The medication of Hsp expression facilitator, as long as there is no particular limitation in HA oligose performance Hsp expresses the scope of reinforced effects, for example injection (intravenously, intramuscular, subcutaneous, intracutaneous, intraperitoneal etc.), intranasal, per os, through skin, suction etc.And, suitable formulation be can select according to such medication, injection (dissolving the solid agent of usefulness etc. when solution, suspension liquid, emulsion, use), tablet, capsule, liquor, granule, powder, fat agent, ointment, plaster, lotion, paste, patch, gelifying agent, seat agent, external pulvis, spray, suction powder etc. for example can be adopted.
There is no particular limitation for the content of the HA oligose in the Hsp expression facilitator (particularly HA4 sugar), when still for example the Hsp expression facilitator being provided as injection, and preferred 0.1～10% (w/v).
When for example the Hsp expression facilitator provided as injection, its form can be any in solution, scars or the lyophilized products.With its filling, be sealed in the proper container such as narrow-necked bottle, small vials, syringe, with the circulation of this form or preserve, can be used as injection and carry out administration.
As mentioned above, as the effective constituent of Hsp expression facilitator, preferred especially HA4 sugar, but in addition, contain the also not influence of HA oligose of other molecular sizes.And among the embodiment from behind as can be seen, HA4 sugar has special extremely significant Hsp and expresses enhancement, so only be increased in the HA4 sugar content in the Hsp expression facilitator, just good effect can be obtained, and the dosage of keeping with the HA oligose same effect of other molecular sizes can be reduced.Thereby the HA oligose in the Hsp expression facilitator especially preferably in fact only is made of 4 sugar, does not preferably add HA4 sugar HA oligose in addition.
(2) administration object of Hsp expression facilitator etc.
Expectation Hsp expression facilitator, a lot of diseases in the disease that cell obstacle that defense reaction hinted that Hsp is produced or necrocytosis etc. cause can tell on, heart disease (myocardial infarction etc.) for example, urine tubule obstacle, the causing circulatory disease, disease of brain (cerebral apoplexy etc.), the ischemic disease that angiostenosises such as sacred disease or ischemic cause, the infull syndromes of acquired immunity, the obstacle of the thymocyte that the administration of immunosuppressor or carcinostatic agent causes, the minimizing of tip T cell, immune related disease such as disease of immune deficiency, hepatitis, inflammation such as ulcerative colitis, wound, microbism, viral infectious disease, alzheimer's disease, diabetes, loose disease, mucocutaneous lymphnode syndrome, schizophrenia, heating, metabolic trouble, cancer etc.
And be not only the disease that cell obstacle or necrocytosis cause, for the cell of expectation Hsp, the object of organization protection's effect, also can be suitable for the Hsp expression facilitator.For this point, in " cell, organization protection's agent " of back, describe in detail.
Given animal, preferred vertebrates, special preferred mammal, preferred especially people with the Hsp expression facilitator.Can be with the prevention of these diseases, the inhibition of carrying out (preventing to worsen), the improvement of symptom or treatment etc. are used the Hsp expression facilitator for purpose, but preferably carry out administration as prophylactic agent.
The use level of HA oligose, particularly HA4 sugar, single administration amount, dosing interval etc. in the Hsp expression facilitator, the medication of toughener, administration form, application target etc., should determine according to patient's concrete symptom, age, sex, body weight etc. are indivedual, there is no particular limitation, but as the clinical administration amount of HA4 sugar, the illustration 300～7500mg of 1 people that is grown up.And Hsp expression enhanced dosing interval, can be once-a-day, also can be divided in 1st 2～3 times.
The Hsp expression facilitator also can be as expressing the relevant experiment test drug of enhancement with stress protein.
[2] cell death inhibitor
Cell death inhibitor is an one of purposes of utilizing the expression enhancement of Hsp.That is, HA oligose (particularly HA4 sugar) not only has Hsp and expresses enhancement, in fact also brings into play the necrocytosis restraining effect, so be applied to the purpose that necrocytosis suppresses.
Can be suitable for the disease of this cell death inhibitor, so long as can suppress the necrocytosis disease by the expression enhancing of Hsp, then there is no particular limitation, the disease that the explanation of Hsp expression facilitator has preferably exemplified.
Given the use level, dosage, dosing interval of animal with this cell death inhibitor, administration purpose, HA oligose (particularly HA4 sugar) etc., identical with the Hsp expression facilitator.
[3] cell obstacle inhibitor
HA oligose (particularly HA4 sugar) not only has the expression enhancement of Hsp, in fact also brings into play cell obstacle restraining effect, so be applied to the purpose that the cell obstacle suppresses.
Can be suitable for the disease of this cell obstacle inhibitor, so long as can suppress the cell disorder disease by the expression enhancing of Hsp, then there is no particular limitation, the disease that the explanation of Hsp expression facilitator has preferably exemplified.
Given the use level, dosage, dosing interval of animal with this cell obstacle inhibitor, administration purpose, HA oligose (particularly HA4 sugar) etc., identical with the Hsp expression facilitator.
[4] cell, organization protection's agent
HA oligose (particularly HA4 sugar) not only has the expression enhancement of Hsp, in fact also brings into play cell, organization protection's effect, so be applied to the purpose of cell, organization protection.
This cell, organization protection's agent go for expecting the cell of Hsp, the object of organization protection's effect.For example, need can be used to cell, organization protection disease etc., or take out the outer cell of organism, organization protection etc.
Disease as needing the cell or tissue protection can exemplify peptide ulceration (stomach, duodenal ulcer etc.), gastritis, hepatopathy (hepatitis etc.), ulcerative colitis, ischemia heart disease, myocarditis, cerebral apoplexy, cerebral infarction etc.
This cell, organization protection's agent specifically, are preferably used as following preparation.
(1) ulcer treatment agent, hepatopathy treatment agent
Aforementioned cell, organization protection's agent by as the preparation of disposing ulcer, can be used as the ulcer treatment agent, by as the preparation of disposing hepatopathy, can be used as the hepatopathy treatment agent.In addition, the implication of " disposal " is described in the present invention, with the prevention of disease, keep (preventing to worsen), alleviate (improvement of symptom) and treatment be a purpose, carries out administration for the animal that suffers from this disease, and " treatment agent " is the medicament that is used for this disposal.
(2) IL-10 produces promotor, IL-8 produces inhibitor
Aforementioned cell, organization protection's agent can be used for IL-10 and reduce the disease cause, maybe need to promote to produce the disease of IL-10 etc.By cell, organization protection's agent are used as the preparation that promotes to produce IL-10, can be used as IL-10 and produce promotor.
And, this cell, organization protection's agent, can be used for IL-8 increases the disease cause, maybe needs to suppress to produce the disease of IL-8 etc.By cell, organization protection's agent are used as the preparation that suppresses to produce IL-8, can be used as IL-8 and produce inhibitor.
As the animal of the administration object of this preparation, or the administration purpose etc., identical with the explanation of Hsp expression facilitator.
(3) organ preservatives
Aforementioned cell, organization protection's agent can be used to protect the cell that takes out outside the organism, the purpose of tissue.Preserve the preparation that takes out the outer cell or tissue of organism by this cell, organization protection's agent are used as, can be used as for example organ preservatives.Specifically, by being to transplant the organ (liver, kidney, heart, lung etc.) take out with this organ preservatives perfusion, or preservation in this organ preservatives, maintenance etc., can suppress cell, the tissue injury (edema, necrocytosis etc.) of this organ.
HA oligose use level in cell, organization protection's agent, single administration (use) amount, dosing interval etc. can be determined individually according to concrete situations such as administration (use) method, form, purposes.
When for example cell, organization protection's agent being produced promotor, IL-8 generation inhibitor etc. to people's administration as ulcer treatment agent, hepatopathy treatment agent, IL-10; as the clinical administration amount of HA oligose (particularly HA4 sugar), can exemplify 120～6000mg of each adult.
With this cell, organization protection's agent, as protection take out the outer cell of organism, when organizational goal is used, can with its as liquor, perfusion with agent, the solid agent etc. of dissolving usefulness and preparationization when using.When using,, can determine the use level of HA oligose (particularly HA4 sugar) individually according to concrete situations such as the organ size of preserving or keep, shelf time, temperature as the organ preservatives.
Also there is no particular limitation for the concentration of HA oligose in this cell, the organization protection's agent (particularly HA4 sugar), but when providing as injection (solution) for example or the liquor of preserving organ, preferred 1ng/ml～1mg/ml.
When cell, organization protection's agent provided as injection or liquor, its form can be any in solution, scars or the lyophilized products.With its filling, be sealed in the proper container such as narrow-necked bottle, small vials, syringe, with the circulation of this form or preserve, can be used as injection and carry out administration.
And then; the present invention not only comprises above-mentioned preparation, is also included within vitro or HA oligose (particularly HA4 oligose) is acted on applicable objects such as cell or bio-tissue in vivo, strengthens the expression of Hsp in this applicable object; suppress necrocytosis; suppress the cell obstacle, or protect cell, tissue (for example, to preserve organ; dispose ulcer; dispose hepatopathy, promote IL-10 to produce, or suppress IL-8 and produce) method.
Description of drawings
Fig. 1 is the elution curve of expression HA4 in gel filtration chromatography.The longitudinal axis of epimere represents that 210nm absorbs, and the longitudinal axis of hypomere represents that RI absorbs.And transverse axis is represented elution time all the time.
Fig. 2 is the elution curve of expression HA6 in gel filtration chromatography.The explanation of the longitudinal axis and transverse axis is identical with Fig. 1.
Fig. 3 is the elution curve of expression HA8 in gel filtration chromatography.The explanation of the longitudinal axis and transverse axis is identical with Fig. 1.
Fig. 4 is the elution curve of expression HA10 in gel filtration chromatography.The explanation of the longitudinal axis and transverse axis is identical with Fig. 1.
Fig. 5 is the elution curve of expression HA12 in gel filtration chromatography.The explanation of the longitudinal axis and transverse axis is identical with Fig. 1.
Fig. 6 is the elution curve of expression HA14 in gel filtration chromatography.The explanation of the longitudinal axis and transverse axis is identical with Fig. 1.
Fig. 7 is the elution curve of expression HA4 in anion-exchange chromatography.The longitudinal axis represents that 210nm absorbs, and transverse axis is represented elution time.
Fig. 8 is the elution curve of expression HA6 in anion-exchange chromatography.The explanation of the longitudinal axis and transverse axis is identical with Fig. 7.
Fig. 9 is the elution curve of expression HA8 in anion-exchange chromatography.The explanation of the longitudinal axis and transverse axis is identical with Fig. 7.
Figure 10 is the elution curve of expression HA10 in anion-exchange chromatography.The explanation of the longitudinal axis and transverse axis is identical with Fig. 7.
Figure 11 is the elution curve of expression HA12 in anion-exchange chromatography.The explanation of the longitudinal axis and transverse axis is identical with Fig. 7.
Figure 12 is the elution curve of expression HA14 in anion-exchange chromatography.The explanation of the longitudinal axis and transverse axis is identical with Fig. 7.
Figure 13 is the elution curve of expression HA48 in anion-exchange chromatography.The explanation of the longitudinal axis and transverse axis is identical with Fig. 7.
Figure 14 is the elution curve of expression HA50 in anion-exchange chromatography.The explanation of the longitudinal axis and transverse axis is identical with Fig. 7.
Figure 15 is the elution curve of expression HA52 in anion-exchange chromatography.The explanation of the longitudinal axis and transverse axis is identical with Fig. 7.
Figure 16 is the figure of the electrophoresis result of expression fluorescent mark oligose.
Figure 17 is the mass spectral figure of expression HA4.
Figure 18 is the mass spectral figure of expression HA6 (epimere), HA8 (stage casing) and HA10 (hypomere).
Figure 19 is the Deco result's of expression HA6 (epimere), HA8 (stage casing) and HA10 (hypomere) figure.
Figure 20 is the mass spectral figure of expression HA12 (epimere), HA14 (stage casing) and HA16 (hypomere).
Figure 21 is the Deco result's of expression HA12 (epimere), HA14 (stage casing) and HA16 (hypomere) figure.
Figure 22 is the mass spectral figure of expression HA48 (epimere), HA50 (stage casing) and HA52 (hypomere).
Figure 23 is the Deco result's of expression HA43 (epimere), HA50 (stage casing) and HA52 (hypomere) figure.
Figure 24 is for expression HA4's 1H-NMR spectrographic figure.
Figure 25 is for expression HA4's 13C-NMR spectrographic figure.
Figure 26 is for expression HA6's 1H-NMR spectrographic figure.
Figure 27 is for expression HA6's 13C-NMR spectrographic figure.
Figure 28 is for expression HA8's 1H-NMR spectrographic figure.
Figure 29 is for expression HA8's 13C-NMR spectrographic figure.
Figure 30 is for expression HA10's 1H-NMR spectrographic figure.
Figure 31 is for expression HA10's 13C-NMR spectrographic figure.
Figure 32 is for expression HA12's 1H-NMR spectrographic figure.
Figure 33 is for expression HA12's 13C-NMR spectrographic figure.
Figure 34 is for expression HA14's 1H-NMR spectrographic figure.
Figure 35 is for expression HA14's 13C-NMR spectrographic figure.
Figure 36 is for expression HA16's 1H-NMR spectrographic figure.
Figure 37 is for expression HA16's 13C-NMR spectrographic figure.
Figure 38 is for expression HA48's 1H-NMR spectrographic figure.
Figure 39 is for expression HA50's 1H-NMR spectrographic figure.
Figure 40 is for expression HA52's 1H-NMR spectrographic figure.
Figure 41 is the HSFl activatory figure that HA4 sugar causes after the expression thermal shocking.
Figure 42 is the figure of the expression of HA4 sugar inductive Hsp72 after the expression thermal shocking.
Figure 43 is for representing that various HA oligose are to the figure by the inhibition degree that lacks serum inductive necrocytosis (apoptosis).
Figure 44 is the figure of expression HA4 sugar for the inhibition effect of organ edema.
Figure 45 is the figure of expression HA4 sugar for the spissated inhibition effect of the chromatin of cell.
Figure 46 is the figure of expression by the minimizing TUNEL staining cell number of HA4 sugar acquisition.
Figure 47 is the figure of expression HA4 sugar for the inhibition effect of cell, tissue disorder.
Figure 48 is the figure of expression HA4 sugar for the inhibition effect of stomach ulcer.
Figure 49 is the figure of expression HA4 sugar to the inhibition effect (GOT activity) of hepatopathy.
Figure 50 is the figure of expression HA4 sugar to the inhibition effect (white blood cell count) of hepatopathy.
Figure 51 promotes the figure of the effect that IL-10 produces for expression HA4 sugar.
Figure 52 suppresses the figure of the effect of IL-8 generation for expression HA4 sugar.
Embodiment
Below, be described more specifically the present invention by embodiment.
Below, HA4 sugar fraction slightly is designated as " HA4 ", HA6 sugar fraction slightly is designated as " HA6 ", HA8 sugar fraction slightly is designated as " HA8 ".
[embodiment 1]
The preparation of oligose of the present invention and fraction of the present invention and physico-chemical property
1. the preparation of oligose of the present invention and fraction of the present invention
With separate from cockscomb, the sodium salt of the HA of purifying is raw material, prepared oligose of the present invention and fraction of the present invention by the following method.In addition, sodium salt as the HA of raw material, by using the electrophoresis (electrophoretic buffer: pyridine-formic acid damping fluid of cellulose acetate membrane, electric current: 15mA, electrophoresis time: 30 minutes) shows single bands of a spectrum, do not detect HA mucopolysaccharide (chrondroitin, sulfuric acid-4-chrondroitin, sulfuric acid-6-chrondroitin, chondroitin sulfate E, chondroitin sulfate D, heparin, heparitin sulfate, dermatan sulfate) in addition.
(preparation example 1) utilizes the decomposition of Unidasa
With the sodium salt 25g of HA be dissolved in contain 0.15M NaCl 0.1M phosphoric acid buffer (pH5.3) 1.IL in.In gained solution, add Unidasa (5.342 units/mg from bull testis; Seikagaku Kogyo Co. Ltd. makes) 200mg, reacted 9 hours down at 37 ℃.
After the reaction, with 10, the 000rpm centrifugation was reclaimed supernatant liquor after 30 minutes.With the supernatant liquor that reclaims, be loaded into Dowex 1 * 2 (100～200 order) (the Dow chemical manufacturing) ion exchange column that has as the strongly basic anionite of the trimethyl ammonium methyl of anionresin base (on the φ 1.5 * 123cm), (wash-out of 0.01M～0.50M) of the straight line concentration gradient by NaCl.By utilizing the uronic acid in the resultant fraction of carbazole method (carbazole method) detection, screened the fraction that contains the HA oligose.Suitable fraction is compiled and concentrated, (Pharmacia makes to utilize Sephadex G-10; φ 3 * 124) carry out desalination, implement freeze-drying afterwards.
The freeze-drying weight of the fraction that obtains is illustrated in the following bracket.
HA4(330mg)、HA6(1210mg)、HA8(305mg)、HA10(1625mg)、HA12(685mg)、HA14(620mg)、HA16(430mg)、HA18(210mg)、HA20(202mg)、HA22(819mg)、HA24(197mg)、HA26(187mg)、HA28(159mg)、HA30(137mg)、HA32(122mg)、HA34(102mg)、HA36(91mg)、HA38(89mg)、HA40(65mg)、HA42(76mg)、HA44(61mg)、HA46(58mg)、HA48(46mg)、HA50(48mg)、HA52(21mg)。
(preparation example 2) utilizes the decomposition of chondroitinase ACI
The sodium salt 8g of HA is dissolved among 0.1M acetate buffer (ph 6.0) 500ml that contains 0.1% bovine serum albumin.
In gained solution, add chondroitinase AC I (Seikagaku Kogyo Co. Ltd.'s manufacturing) 32 units, reacted 6 hours down at 37 ℃.
After the reaction, with 10, the 000rpm centrifugation was reclaimed supernatant liquor after 30 minutes.With the supernatant liquor that reclaims, be loaded into Dowex 1 * 2 (100～200 order) (Dow chemical manufacturing) ion exchange column (on the φ 4.5 * 123cm), (wash-out of 0.01M～0.50M) of the straight line concentration gradient by NaCl.By utilizing the uronic acid in the carbazole method detection gained fraction, screened the fraction that contains the HA oligose.Concentrate after suitable fraction compiled, (Pharmacia makes to utilize Sephadex G-10; φ 3 * 124) carry out desalination, implement freeze-drying afterwards.
The freeze-drying weight of gained fraction is illustrated in the following bracket.Wherein the non-reduced terminal sugar of [Δ] expression is unsaturated sugar.
ΔHA4(133mg)、ΔHA6(133mg)、ΔHA8(84mg)、ΔHA10(109mg)、ΔHA12(100mg)、ΔHA14(101mg)、ΔHA16(73mg)、ΔHA18(31mg)、ΔHA20(9mg)。
(preparation example 3) DMSO method
The sodium salt 10g of HA is dissolved among dimethyl sulfoxide (DMSO) (DMSO) 3L that contains 10% 0.1M HCl.105 ℃ of following heat treated 16 hours.
After the processing, be loaded into Dowex 1 * 2 (100～200 order) (Dow chemical manufacturing) ion exchange column and (carried out stratographic analysis on the φ 3.0 * 78cm).Wash-out is by the straight line concentration gradient (0.01M～0.50M) carry out of NaCl.By utilizing the carbazole method to detect uronic acid in the resultant fraction, screened the fraction that contains the HA oligose.Concentrate after suitable fraction compiled, (Pharmacia makes to utilize Sephadex G-10; φ 3 * 124) carry out desalination, implement freeze-drying afterwards.
The freeze-drying weight of the fraction that obtains is illustrated in the following bracket.
HA2(50mg)、HA4(1100mg)、HA6(232mg)、HA8(1015mg)、HA10(1033mg)、HA12(459mg)。
With the HA oligose of all size that obtains like this, following various analyses have been carried out.
2. the physico-chemical property of fraction of the present invention
Each HA oligose (sodium salt) fraction for obtaining in the above-mentioned preparation example 1 has detected various physico-chemical properties.
(1) gel filtration chromatography analysis
The post that uses: TSKGe12500+3000+4000pwxl (TOSOH Co., Ltd.)
Solvent: 0.05M NaCI
Flow velocity: 0.6ml/ branch
Detect wavelength: 210nm, reach differential refractometer (RI)
Sample heap(ed) capacity:, be 200 μ g/ emissions (Shot) as the amount of HA oligose
Elution curve when using HA4 (the 1st crowd)～HA14 as sample is illustrated among Fig. 1～Fig. 6.The epimere of each figure is illustrated in the result that 210nm detects, and hypomere is represented the result with the differential refractometer detection.
From these figure, any as can be seen fraction is all expressed single in fact peak.
And, as shown in table 1 with respect to the relative area at the main HA peak of the peak area sum of whole HA oligose in the fraction, be more than 85% during based on the 210nm absorption detecting, be more than 98% when detecting with differential refractometer (RI).
Table 1
Sample With respect to the area of the main peak at whole peaks than (%)
210nm RI
HA4 (the 1st crowd) 89.037 99.854
HA6 98.573 99.815
HA8 96.617 98.929
HA10 98.594 99.287
HA12 100.000 100.000
HA14 98.334 99.796
(2) Analysis by Chromatography
Post: YMC NH2 post (YMC of Co., Ltd.)
Solvent: use NaH 2PO 4The concentration gradient of 0M to 0.8M
Flow velocity: 1ml/ branch
Detect wavelength: 210nm
Sample heap(ed) capacity:, be 20 μ g/ emissions as the amount of HA oligose
Elution curve when using HA4 (the 1st crowd)～HA14 and HA48～HA52 as sample is illustrated among Fig. 7～Figure 15.From these figure, any as can be seen fraction is all expressed single in fact peak.And, with respect to the relative area at the main HA peak of the peak area sum of whole HA oligose in the fraction, as shown in table 2 being more than 90%.
Table 2
Sample With respect to the area of the main peak at whole peaks than (%)
HA4 (the 1st crowd) HA6 HA8 HA10 HA12 HA14 HA48 HA50 HA52 98.5007 97.9140 98.1209 97.4236 94.5425 94.7181 94.4743 93.4305 94.2492
(3) fluorescent mark gel electrophoresis analysis
HA4, HA6, HA8, HA10, HA12, HA14 and HA16 are carried out following processing separately.Simultaneously, (also contain HA2 sugar for HA oligosaccharide mixture fraction; Below slightly be designated as " mixture ") also carried out same processing.
(3-1) modulation of fluorescent mark oligose
With the contained HA oligose of each fraction, use FACE R(Novato CA.U.S.A) has implemented fluorescent mark to N-linked OligosaccharideProfiling Kit for Glyko, Inc..
To contain about 2nmol (for mixture, with the HA2 sugar about 2nmol of unit) fraction of HA oligose amount, in the 0.5mL plastics tubing, utilize the centrifugal vacuum drier (Speedvac, AS160, SAVANT INSTRUMENTS INC.NY.U.S.A) that freezes to carry out drying.After the drying, respectively add the 8-amino naphthalenes-1,3 of 5 μ L in each pipe, 6-trisulfonic acid disodium salt (ANTS) solution (GLYKO, L2, Part#50058:reconstituted OLIGO LabelingDye) stirs the back and placed 15 minutes in room temperature.And then each adds the cyanogen boron hydrogen sodium solution (GLYKO, L1, Part#50056:Labeling Reducing Agent) of 5 μ L, sealing and 36 ℃ of following incubations 16 hours.
After the incubation, passed through 15 minutes on the vacuum drier, carry out the part drying, add 20 μ L distilled water afterwards centrifugal freezing.
(3-2) electrophoresis of fluorescent mark oligose
Use GLYKO O-linked Oligsaccharide Profiling Gel (GLYKO, Part#60200; Degree of crosslinking is 36% polyacrylamide gel) carried out electrophoresis.
(GLYKO Part#7000) makes it reach 1.5L with dissolved in distilled water, and cools off in ice with 1 bag OLIGO Gel Running Buffer.
(Part#40026) middle recirculated cooling water adds chilled Running Buffer, and puts gel gel box therein for GLYKO, GLYKO Gel Box.Gel box also cools off with ice.
Respectively get the various fraction solution that 2 μ L contain fluorescently-labeled HA oligose, with distilled water 3 μ L and Loading Buffer (GLYKO, Part#50064) 5 μ L mixing.Getting 2 μ L mixture solutions mixes with Loading Buffer 2 μ L.These 4 μ L separately are loaded on the gel.The heap(ed) capacity of each fraction is that to make the heap(ed) capacity of the HA oligose of each swimming lane be 80pmol.
With the constant voltage electrophoresis of 1000V 160 minutes, by ultraviolet transilluminator (NLM-20E type, UVP, CA, U.S.A) fluorescence that sends of light of irradiation 365nm utilizes fast imaging photographic camera (MAMIYA, Professional SD, f=127mm) and fast imaging egative film (Polaloid RPolapan T667, iso3000, Polapoid corp.MA.U.S.A.) carry out record.Its result is illustrated among Figure 16.
The relation of swimming lane and sample is as follows among Figure 16.
Swimming lane 1: mixture, swimming lane 2:HA4, swimming lane 3:HA6, swimming lane 4:HA8, swimming lane 5:HA10, swimming lane 6:HA12, swimming lane 7:HA14, swimming lane 8:HA16.
Mixture shows ladder-like spectrum belt, but each oligose fraction of fractionated, all forms the single bands of a spectrum that have corresponding to its big or small swimming degree, and, do not detect the bands of a spectrum of other big or small HA oligose.
In addition, size is more little, and the swimming degree that obtains is big more.The position of oligose bands of a spectrum in each fraction (swimming position), each big or small position of the ladder-like spectrum belt of expressing with mixture is almost consistent, even in the mixture of the oligose of a plurality of sizes, the swimming degree of each molecule is not affected yet.
GLYKO O-linked Profiling Gel is that degree of crosslinking is 36% polyacrylamide gel.8-amino naphthalenes-1,3,6-trisulfonic acid disodium salt (ANTS) is the fluorescent substance with trivalent negative charge.
During as fluorescent substance, all oligose on this gel are all motionless substantially with uncharged 2-amino acridones (AMAC).Thereby the negative charge deficiency of HA oligose itself only in the electrophoresis of this high-crosslinking-degree gel needs the electric charge of bonded ANTS as can be seen.And, do not use known and the interactional boric acid of sugar chain skeleton structure, so the interaction of the mesh size of the size of hint oligose and polyacrylamide influences the swimming degree.
Use other gels (degree of crosslinking is 20% or 21%) of Glyko company, and during with the ANTS mark, can not separate below the HA8 sugar, or HA4 sugar is following can not detect with the preceding line overlap of swimming.
(4) mass spectroscopy
By electro-spray ionization mass spectrometry (electrospray ionization massspectrometry; ESIMS) carry out mass spectroscopy.
(4-1) method
(4-1-1) analytical sample
HA4 (the 1st crowd), HA6, HA8, HA10, HA12, HA14, HA16, HA48, HA50 and HA52 are made into the aqueous solution, and making separately, concentration reaches 2.6mg/ml, 1.2mg/ml, 1.4mg/ml, 3.0mg/ml, 2.0mg/ml, 6.5mg/ml, 1.3mg/ml, 1mg/ml, 1mg/ml and 1mg/ml successively.
In addition, for the theoretical value of the molecular weight relevant with the HA oligose, having structure shown in the following formula (1) with HA4 sugar～52 sugar is that try to achieve on the basis.
Formula (1)
Figure C0181243200341
(in the formula, n represents 1～29 integer, and M represents proton or monovalent cation, and Ac represents ethanoyl)
(4-1-2) reagent
Methyl alcohol of Shi Yonging (with the pure medicine of light) and distilled water (with the pure medicine of light) are used for HPLC in test, and ammonium formiate (with the pure medicine of light) has used superfine.
(4-1-3) instrument and equipment
1) HPLC system
Agilent 1100 series: binary pump, de-gassing vessel, self-actuated sampler (AgilentTechnologies)
2) mass spectrograph
Triple quadrupole type mass spectrograph: TSQ (ThermoQuest)
(4-1-4) ESIMS analysis condition
Importing sample to mass spectrograph is to inject each sample solution 5 μ L in by the HPLC system on being connected mass spectrograph or 10 μ L carry out.The analysis condition of HPLC and ESIMS is as follows.
1)HPLC
Mobile phase: the 10mM ammonium formiate aqueous solution/methyl alcohol=80/20
Flow velocity: 0.2ml/min
Post: do not have
Injection rate: 10 μ L
2)ESIMS
Probe: Off-axis
Ion mode: negatively charged ion pattern
Ionization method: ESI method (electro-spray ionization method)
ESI spray voltage: 4.5kV
Hot capillary temperature: 350 ℃
Assist gas: 35 units
Shielding gas (sheath gas): 50psi
Sweep limit: m/z 10-2500 or 10-4000
Sweep time: 1.5 seconds or 3 seconds
(4-1-5) Deco analysis
From the Deco analysis of the ESIMS spectrum that observes estimation molecular weight, undertaken by analysis software Xcalibur Bioworks (ThermoQuest).
(4-2) result
The negatively charged ion ESIMS spectrometry result of HA4～HA16 is summarised in table 3 and the table 4, and the spectral representation of HA4～HA16 or HA48～HA52 is in Figure 17～Figure 23.
Table 3
Sample Intend molion and relevant ions (m/z) thereof The Deco quality
[M-H] - [M-2H] 2- [M-3H] 3- [M-4H] 4- [M-5H] 5- [M+Na- 2H] - [M+Na- 3H] 2- [M+2Na- 4H] 2- [M+Na- 4H] 3- [M+2Na- 5H] 3- [M+3Na- 6H] 3- [2M-H] - [2M- 3H] 3-
HA4 (the 1st crowd) 774.9 100 386.9 55 797.1 27 1551.6 15
HA6 1153.9 45 576.6 100 383.8 1 1176.1 2 587.6 19 769.1 3 11551
HA8 1533.9 36 766.1 100 510.3 3 777.0 9 1022.3 10 1534.5
HA10 1912.8 16 955.9 100 636.9 15 967.0 4 1275.2 9 1913.7
HA12 2292.1 12 1145.5 100 763.2 91 572.3 13 1156.3 16 1167.4 8 770.5 15 777.9 8 1527.9 11 2293.1
HA14 13353 79 889.5 100 666.6 12 1346.2 9 1357.5 4 897.1 14 1780.6 4 2671.9
HA16 1524.8 52 1016.1 100 761.6 17 608.9 5 1535.7 4 1023.4 16 1030.6 9 1037.6 3 3051.2
Epimere: observed value
Hypomere: the relative intensity at peak (%)
Table 4
Sample Molecule associate ion (m/z) The Deco quality
[M-5H] 5- [M-6H] 6- [M-7H] 7- [M-8H] 8- [M-9H] 9- [M- 10H] 10- [M- 11H] 11- [M- 12H] 12- [M- 13H] 13- [M- 14H] 14- [M+Na- 6H] 5- [M+2Na -7H] 5- [M+3Na -8H] 5-
HA48 1822.4 100 1518.7 7 1301.9 6 1138.4 12 1011.7 16 910.5 37 827.9 77 758.6 59 700.2 22 650.0 8 1826.7 45 1831.7 25 1835.6 23 9117.2
HA50 1898.2 100 1581.4 7 1355.5 2 1186.5 7 1054.1 8 948.7 16 861.9 26 790.3 23 729.4 19 677.1 14 1903.0 43 1907.3 19 1911.8 12 9496.1
HA52 1974.2 100 1645.3 23 1409.5 7 1233.0 11 1096.4 23 986.7 27 896 69 821.8 98 758.4 45 704.3 20 1979.3 50 1983.5 37 1987.4 39 9875.0
Epimere: observed value
Hypomere: the relative intensity at peak (%)
In above result, observed the ion relevant with various molecules that all is considered to from the HA oligose, this and HA4 (the 1st crowd) are equivalent to HA4 sugar, and HA6 is equivalent to HA6 sugar, HA8 is equivalent to HA8 sugar, HA10 is equivalent to HA10 sugar, and HA12 is equivalent to HA12 sugar, and HA14 is equivalent to HA14 sugar, HA 16 is equivalent to HA16 sugar, HA48 is equivalent to HA48 sugar, and HA50 is equivalent to HA50 sugar, and the HA52 explanation contradiction not that is equivalent to HA52 sugar.
According to above result, the theoretical value of single isotopic molecule amount is set at 1 o'clock, has tried to achieve the relative value of the measured value of main peak.Its result is illustrated among the 5A.In addition, used relevant [M-H] for HA4 -Measured value and theoretical value.
Table 5A
Sample Measured value Theoretical value Relative value
HA4 (the 1st crowd) 774.9 775.2 0.9996
HA6 1155.1 1155.3 0.9998
HA8 1534.5 1534.5 1.0000
HA10 1913.7 1913.6 1.0001
HA12 2293.1 2292.7 1.0002
HA14 2671.9 2671.8 1.0000
HA16 3051.2 3050.9 1.0001
HA48 9117.2 9116.7 1.0001
HA50 9496.1 9495.8 1.0000
HA52 9875.0 9874.9 1.0000
In addition, the theoretical value of molecular-weight average is set at 1 o'clock, has tried to achieve the relative value of the measured value of main peak.Its result is illustrated among the 5B.In addition, used relevant [M-H] for HA4 -Measured value and theoretical value.
Table 5B
Sample Measured value Theoretical value Relative value
HA4 (the 1st crowd) 774.9 775.6 0.9991
HA6 1155.1 1156.0 0.9992
HA8 1534.5 1535.3 0.9995
HA10 1913.7 1914.6 0.9995
HA12 2293.1 2293.9 0.9997
HA14 2671.9 2673.3 0.9995
HA16 3051.2 3052.6 0.9995
HA48 9117.2 9121.7 0.9995
HA50 9496.1 9501.0 0.9995
HA52 9875.0 9880.3 0.9995
These presentation of results constitute the single isotopic molecule amount of HA oligose of fraction of the present invention or the theoretical value of molecular-weight average and are set at 1 o'clock, and the mass spectroscopy by fraction obtains measured value all in the scope of 0.999～1.001 (relative value).
(5) ultimate analysis
With each comfortable 80 ℃ of following drying under reduced pressure of HA4 (the 1st crowd), HA6, HA8, HA10, HA12, HA14 and HA16 2 hours, measure with elemental analyzer immediately.Its result is illustrated in the table 6.
Table 6
Sample Element Theoretical value (%) Measured value (%) Error
HA4 (the 1st crowd) C H N Na 40.98 5.16 3.41 5.60 40.63 5.20 3.34 5.48 0.35 -0.04 0.07 0.12
HA6 C H N Na 41.28 5.11 3.44 5.64 40.74 5.17 3.31 5.75 0.54 -0.06 0.13 -0.11
HA8 C H N Na 41.44 5.09 3.45 5.67 41.41 5.22 3.36 5.41 0.03 -0.13 0.09 0.26
HA10 C H N Na 41.53 5.08 3.46 5.68 41.41 5.24 3.43 5.45 0.12 -0.16 0.03 0.23
HA12 C H N Na 41.59 5.07 3.46 5.69 40.96 5.33 3.23 5.41 0.63 -0.26 0.23 0.28
HA14 C H N Na 41.64 5.06 3.47 5.69 41.44 5.36 3.37 5.20 0.20 -0.30 0.10 0.49
This presentation of results, the theoretical value (weight %) of the ratio of content separately of carbon (C), hydrogen (H), nitrogen (N) and sodium (Na) in the HA oligose (sodium salt) of formation fraction of the present invention, the measured value (weight %) of each element that obtains by ultimate analysis with this fraction poor is all in ± 1 (weight %) scope.
(6) NMR (Nuclear Magnetic Resonance) spectrum (NMR)
Utilize VARIAN UnityInova500 type to measure HA oligomeric carbon fraction 1H-NMR reaches 13C-NMR.Measure solvent and used D 2O.In be designated as t-BuOH ( 1H:1.23ppm, 13C:32.461ppm), measure temperature and be set at 23 ℃, measure.The result of each HA oligose fraction is as follows.In addition, spectral representation separately is in Figure 24～Figure 40.
(6-1) measurement result of HA4 (group 1)
500MHz 1H-NMR δ；
2.004(s，3H，NAc)，2.018，2.019(3H，NAc)，3.292-3.326(m，1H，H-2d)，3.354(dd，1H，J 1，2＝7.9Hz，J 2，3＝9.5Hz，H-2b)，4.027(dd，0.6H，J 1，2＝3.5Hz，J 2，3＝10.6Hz，H-2aα)，4.453(d，J 1，2＝7.9Hz，H-1d)，4.460(d，H-1bβ)，4.502(d，0.6H，H-1bα)，4.555(d，1H，J 1，2＝8.4Hz，H-1c)，4.705(d，0.4H，J 1，2＝8.4Hz，H-1aβ)，5.144(d，0.6H，H-1aα)
As the spectral representation of analysis foundation in Figure 24.
125MHz 13C-NMRδ；
24.84,25.09,25.36 (NHCOCH 3), 55.81 (C-2a α), 57.09 (C-2c), 58.44 (C-2a β), 63.40,63.57 (C-6a or C-6c),
75.29,75.33 (C-2b α or C-2b β), 75.58 (C-2d), 93.92 (C-1a α), 97.61 (C-1a β), 103.44 (C-1c),
105.80,105.84,105.95 (C-1b α or C-1b β or C-1d),
177.00,177.04,177.41,177.68,177.80,178.33 (carbonyl)
As the spectral representation of analysis foundation in Figure 25.
These results are with the contradiction not of the structure (structure of β-D-glucopyranosyl uronic acid sodium-(1 3)-2-acetamido-2-deoxidation-β-D-glucopyranosyl-(1 4)-(β-D-glucopyranosyl uronic acid sodium)-(1 3)-2-acetamido-2-deoxidation-D-pyranoglucose) represented of n=1 in the preceding formula (1).
(6-2) measurement result of HA6
500MHz 1H-NMR δ；
2.002,2.010,2.017 (9H, NAc), 3.291-3.368 (m, 3H, H-2b, H-2d and H-2f), 4.026 (dd, 0.6H, J1,2=3.5Hz, J2,3=10.6Hz, H-2a α), 4.452,4.459 (d * 2,2.4H, J1,2=7.8Hz, J1,2=7.8Hz, H-1b β, H-1d and H-1f), 4.500 (d, 0.6H, J1,2=7.9Hz, H-1b α)
4.544,4.550 (d * 2,2H, J 1,2=8.5Hz, J 1,2=8.5Hz, H-1c or H-1e), 4.702 (d, J 1,2=8.3Hz, H-1a β), 5.142 (d, 0.6H, H-1a α)
As the spectral representation of analysis foundation in Figure 26.
125MHz 13C-NMRδ；
24.84,25.09,25.35 (NHCOCH 3), 55.81 (C-2a α), 57.09, (57.16 C-2c or C-2e), 58.44 (C-2a β), 63.40, (63.57 C-6a or C-6c or C-6e), 75.28,75.31,75.34,75.57 (C-2b α or C-2b β or C-2d or C-2f), 93.92 (C-1a α), (97.61 C-1a β), 103.40,103.45 (C-1c or C-1e)
105.80,105.85,105.94,106.02 (C-1b α or C-1b β or C-1d or C-1f), 176.97,177.40,177.68,177.80,178.27 (carbonyl)
As the spectral representation of analysis foundation in Figure 27.
These results are with structure (β-D-glucopyranosyl uronic acid sodium-[(1 3)-2-acetamido-2-deoxidation-β-D-glucopyranosyl-(1 4)-(β-D-glucopyranosyl uronic acid sodium)] that represent of n=2 in the preceding formula (1) 2The structure of-(1 3)-2-acetamido-2-deoxidation-D-pyranoglucose) contradiction not.
(6-3) measurement result of HA8
500MHz 1H-NMRδ；
2.002,2.003,2.008,2.016 (12H, NAc), 3.290-3.368 (m, 4H, H-2b, H-2d, H-2f and H-2h),
4.025(dd，0.6H，J 1，2＝3.5Hz，J 2，3＝10.6Hz，H-2aα)，
4.450,4.458 (d * 2,3.4H, J 1,2=7.8Hz, J 1,2=7.8Hz, H-1b β, H-1d, H-1f and H-1h),
4.500(d，0.6H，J 1，2＝7.8Hz，H-1bα)，
4.539,4.543,4.550 (d * 3,3H, J 1,2=8.4Hz, J 1,2=8.5Hz, J 1,2=8.4Hz, H-1c or H-1e or H-1g), 4.702 (d, 0.4H, J 1,2=8.4Hz, H-1a β), 5.142 (d, 0.6H, H-1a α)
As the spectral representation of analysis foundation in Figure 28.
125MHz 13C-NMR δ；
24.84，25.09，25.35(NHCOCH 3)，55.82(C-2aα)，
57.10,57.16 (C-2c, C-2e and C-2g), 58.44 (C-2a β),
63.40,63.57 (C-6a, C-6c and C-6e),
75.28,75.34,75.57 (C-2b, C-2d, C-2f and C-2h),
93.92(C-1aα)，97.61(C-1aβ)，
103.39,103.45 (C-1c, C-1e and C-1g),
105.80,105.85,105.95,106.04 (C-1b or C-1d or C-1f or C-1h), 176.93,177.40,177.68,177.80,178.27 (carbonyl)
As the spectral representation of analysis foundation in Figure 29.
These results are with structure (β-D-glucopyranosyl uronic acid sodium-[(1 3)-2-acetamido-2-deoxidation-β-D-glucopyranosyl-(1 4)-(β-D-glucopyranosyl uronic acid sodium)] that represent of n=3 in the preceding formula (1) 3The structure of-(1 3)-2-acetamido-2-deoxidation-D-pyranoglucose) contradiction not.
(6-4) measurement result of HA10
500MHz 1H-NMRδ；
2.001，2.007，2.016(15H，NAc)，4.025(dd，0.6H，J 1，2＝3.6Hz，J 2，3＝10.6Hz，H-2aα)，
(4.441-4.465 m, H-1b β, H-1d, H-1f, H-1h and H-1j), 4.499 (d, J 1,2=7.8Hz, H-1b α),
4.538,4.549 (d * 2, J 1,2=8.4Hz, J 1,2=8.4Hz, H-1c, H-1e, H-1g and H-1i), 4.701 (d, J 1,2=8.4Hz, H-1a β), 5.141 (d, 0.6H, H-1a α)
As the spectral representation of analysis foundation in Figure 30.
125MHz 13C-NMRδ；
24.84，25.09，25.35(NHCOCH 3)，55.82(C-2aα)，
57.10,57.16 (C-2c, C-2e, C-2g and C-2i), 58.44 (C-2a β),
63.37,63.57 (C-6a, C-6c, C-6e, C-6g and C-6i),
75.35,75.58 (C-2b, C-2d, C-2f, C-2h and C-2j),
93.92(C-1aα)，97.61(C-1aβ)，
103.39,103.46 (C-1c, C-1e, C-1g and C-1i),
105.80,105.86,105.94,106.05 (C-1b, C-1d, C-1f, C-1h and C-1j), 176.94,176.99,177.41,177.68,177.81,178.30 (carbonyl)
As the spectral representation of analysis foundation in Figure 31.
These results are with structure (β-D-glucopyranosyl uronic acid sodium-[(1 3)-2-acetamido-2-deoxidation-β-D-glucopyranosyl-(1 4)-(β-D-glucopyranosyl uronic acid sodium)] that represent of n=4 in the preceding formula (1) 4The structure of-(1 3)-2-acetamido-2-deoxidation-D-pyranoglucose) contradiction not.
(6-5) measurement result of HA12
500MHz 1H-NMRδ；
2.002，2.006，2.016(18H，NAc)，4.025(dd，0.6H，J 1，2＝3.6Hz，J 2，3＝10.7Hz，H-2aα)，
(4.440-4.465 m, H-1b β, H-1d, H-1f, H-1h, H-1j and H-IL), 4.498 (d, J 1,2=7.8Hz, H-1b α),
4.538,4.549 (d * 2, J 1,2=8.4Hz, J 1,2=8.4Hz, H-1c, H-1e, H-1g, H-1i and H-1k), 4.701 (d, J 1,2=8.5Hz, H-1a β), 5.141 (d, 0.6H, H-1a α)
As the spectral representation of analysis foundation in Figure 32.
125MHz 13C-NMRδ；
24.84，25.09，25.35(NHCOCH 3)，55.82(C-2aα)，
57.09,57.16 (C-2c, C-2e, C-2g and C-2i), 58.44 (C-2a β),
(63.37 C-6a, C-6c, C-6e, C-6g and C-6i),
75.35,75.58 (C-2b, C-2d, C-2f, C-2h and C-2j),
93.92(C-1aα)，97.61(C-1aβ)，
103.40,103.45 (C-1c, C-1e, C-1g and C-1i),
105.80,105.86,106.05 (C-1b, C-1d, C-1f, C-1h and C-1j),
176.95,177.40,177.80,178.31 (carbonyls)
As the spectral representation of analysis foundation in Figure 33.
These results are with structure (β-D-glucopyranosyl uronic acid sodium-[(1 3)-2-acetamido-2-deoxidation-β-D-glucopyranosyl-(1 4)-(β-D-glucopyranosyl uronic acid sodium)] that represent of n=5 in the preceding formula (1) 5The structure of-(1 3)-2-acetamido-2-deoxidation-D-pyranoglucose) contradiction not.
(6-6) measurement result of HA14
500MHz 1H-NMRδ；
2.001，2.006，2.016(21H，NAc)，4.024(dd，0.6H，J 1，2＝3.6Hz，J 2，3＝10.6Hz，H-2aα)，
(4.439-4.464 m, H-1b β, H-1d, H-1f, H-1h, H-1j, H-IL and H-1n), 4.498 (d, J 1,2=7.8Hz, H-1b α),
4.537,4.549 (d * 2, J 1,2=8.4Hz, J 1,2=8.3Hz, H-1c, H-1e, H-1g, H-1i, H-1k and H-1m),
4.701(d，J 1，2＝8.4Hz，H-1aβ)，5.141(d，0.6H，H-1aα)
As the spectral representation of analysis foundation in Figure 34.
125MHz 13C-NMRδ；
24.84，25.09，25.34(NHCOCH 3)，55.81(C-2aα)，
57.09,57.15 (C-2c, C-2e, C-2g and C-2i), 58.43 (C-2a β),
63.36,63.57 (C-6a, C-6c, C-6e, C-6g and C-6i),
75.35,75.58 (C-2b, C-2d, C-2f, C-2h and C-2j),
93.91(C-1aα)，97.61(C-1aβ)，
(103.40 C-1c, C-1e, C-1g and C-1i),
105.80,105.85,105.94,106.07 (C-1b, C-1d, C-1f, C-1h and C-1j), 176.95,177.40,177.67,177.80,178.30 (carbonyl)
As the spectral representation of analysis foundation in Figure 35.
These results are with structure (β-D-glucopyranosyl uronic acid sodium-[(1 3)-2-acetamido-2-deoxidation-β-D-glucopyranosyl-(1 4)-(β-D-glucopyranosyl uronic acid sodium)] that represent of n=6 in the preceding formula (1) 6The structure of-(1 3)-2-acetamido-2-deoxidation-D-pyranoglucose) contradiction not.
(6-7) measurement result of HA16
500MHz 1H-NMRδ；
2.002，2.006，2.016(24H，NAc)，4.024(dd，0.6H，J 1，2＝3.6Hz，J 2，3＝10.6Hz，H-2aα)，
(4.434-4.464 m, H-1b β, H-1d, H-1f, H-1h, H-1j, H-IL, H-1n and H-1p), 4.498 (d, J 1,2=7.8Hz, H-1b α),
4.537,4.549 (d * 2, J 1,2=8.4Hz, J 1,2=8.3Hz, H-1c, H-1e, H-1g, H-1i, H-1k, H-1m and H-1o),
4.701(d，J 1，2＝8.4Hz，H-1aβ)，5.141(d，0.6H，H-1aα)
As the spectral representation of analysis foundation in Figure 36.
125MHz 13C-NMRδ；
24.84，25.09，25.35(NHCOCH 3)，55.82(C-2aα)，
57.10,57.16 (C-2c, C-2e, C-2g, C-2i, C-2k, C-2m and C-2o),
58.44(C-2aβ)，
63.37,63.57 (C-6a, C-6c, C-6e, C-6g, C-6i, C-6k, C-6m and C-6o),
75.35,75.58 (C-2b, C-2d, C-2f, C-2h, C-2j, C-2l, C-6n and C-6p), 93.92 (C-1a α), 97.61 (C-1a β),
(103.40 C-1c, C-1e, C-1g, C-1i, C-1k, C-1m and C-1o),
105.80,105.86,105.95,106.08 (C-1b, C-1d, C-1f, C-1h, C-1j, C-IL, C-1n and C-1p),
176.98,177.41,177.68,177.81,178.33 (carbonyls)
As the spectral representation of analysis foundation in Figure 37.
These results are with structure (β-D-glucopyranosyl uronic acid sodium-[(1 3)-2-acetamido-2-deoxidation-β-D-glucopyranosyl-(1 4)-(β-D-glucopyranosyl uronic acid sodium)] that represent of n=7 in the preceding formula (1) 7The structure of-(1 3)-2-acetamido-2-deoxidation-D-pyranoglucose) contradiction not.
(6-8) measurement result of HA48
500MHz 1H-NMRδ；
1.998，2.005，2.016(72H，NAc)，4.024(dd，0.6H，J 1，2＝3.6Hz，J 2，3＝10.6Hz，H-2aα)，
(4.395-4.470 m, GlcA-unit),
4.498(d，J 1，2＝8.0Hz，H-1bα)，
4.537,4.610 (d * 2, J 1,2=8.2Hz, J 1,2=7.8Hz, the GlcNAc-unit),
4.700(d，J 1，2＝8.3Hz，H-1aβ)，5.141(d，0.6H，J 1，2＝3.6Hz，H-1aα)
As the spectral representation of analysis foundation in Figure 38.
These results are with structure (β-D-glucopyranosyl uronic acid sodium-[(1 3)-2-acetamido-2-deoxidation-β-D-glucopyranosyl-(1 4)-(β-D-glucopyranosyl uronic acid sodium)] that represent of n=23 in the preceding formula (1) 23The structure of-(1 3)-2-acetamido-2-deoxidation-D-pyranoglucose) contradiction not.
(6-9) measurement result of HA50
500MHz 1H-NMRδ；
2.005，2.017(75H，NAc)，4.024(dd，0.6H，J 1，2＝3.6Hz，J 2，3＝10.6Hz，H-2aα)，
(4.390-4.470 m, GlcA-unit),
4.498(d，J 1，2＝7.9Hz，H-1bα)，
4.537,4.610 (d * 2, J 1,2=8.1Hz, J 1,2=8.1Hz, the GlcNAc-unit),
4.701(d，J 1，2＝8.4Hz，H-1aβ)，5.141(d，0.6H，J 1.2＝3.6Hz，H-1aα)
As the spectral representation of analysis foundation in Figure 39.
These results are with structure (β-D-glucopyranosyl uronic acid sodium-[(1 3)-2-acetamido-2-deoxidation-β-D-glucopyranosyl-(1 4)-(β-D-glucopyranosyl uronic acid sodium)] that represent of n=24 in the preceding formula (1) 24The structure of-(1 3)-2-acetamido-2-deoxidation-D-pyranoglucose) contradiction not.
(6-10) measurement result of HA52
500MHz 1H-NMRδ；
2.005，2.017(78H，NAc)，4.024(dd，0.6H，J 1，2＝3.6Hz，J 2，3＝10.6Hz，H-2aα)，
(4.375-4.475 m, GlcA-unit),
4.498(d，J 1，2＝8.2Hz，H-1bα)，
4.536,4.610 (d * 2, J 1,2=8.1Hz, J 1,2=8.1Hz, the GlcNAc-unit),
4.701(d，J 1，2＝8.4Hz，H-1aβ)，5.141(d，0.6H，J 1，2＝3.4Hz，H-1aα)
As the spectral representation of analysis foundation in Figure 40.
These results are with structure (β-D-glucopyranosyl uronic acid sodium-[(1 3)-2-acetamido-2-deoxidation-β-D-glucopyranosyl-(1 4)-(β-D-glucopyranosyl uronic acid sodium)] that represent of n=25 in the preceding formula (1) 25The structure of-(1 3)-2-acetamido-2-deoxidation-D-pyranoglucose) contradiction not.
(7) impurity beyond the HA oligose
Measured the content of following impurity in each fraction.
(7-1) protein
Protein content is a reference material with the bovine serum albumin, utilizes BioRad ProteinAssay test kit (manufacturing of BioRad company) to measure.
(7-2)DNA
DNA utilizes threshold method (DNA determinator: Threshold (U.S. BioRad manufacturing)) to measure.
(7-3) intracellular toxin
Intracellular toxin utilizes Toxycolor System (trade(brand)name; Seikagaku Kogyo Co. Ltd. makes) measure by limulus test.
Table 7
Sample Protein (%) DNA Intracellular toxin (pg/ml)
HA4 HA6 HA8 HA10 HA12 HA14 Detect following " " " " of boundary " Detect following " " " " of boundary " 0.5 0.1 0.1 0.7 0.1 0.3
This presentation of results, protein in the fraction of the present invention and the content of DNA are all detecting below the boundary, and endotoxic content can obtain any one and all not contain endotoxic conclusion in fact also for there not being effect in fact.
[embodiment 2]
＜material etc. 〉
(1) substances etc.
At first, the substances of using in the present embodiment is described.
Substances (the contracted notation in the following use bracket.And in following formula, GlcA represents glucuronic acid residue, and GlcNAc represents N-acetyl glucosamine residue, and Gal represents galactose residue, (6S) expression 6-O-sulfuric ester, and-expression glycosidic linkage, Δ is represented unsaturated sugar)
Saturated 2 sugar of HA (HA2)
GlcAβl-3GlcNAc
Unsaturated 2 sugar of HA (Δ HA2)
ΔGlcAβ1-3GlcNAc
Saturated 4 sugar of HA (HA4)
GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAc
Unsaturated 4 sugar of HA (Δ HA4)
ΔGlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAc
Saturated 6 sugar of HA (HA6)
GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAc
Unsaturated 6 sugar of HA (Δ HA6)
ΔGlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAc
Saturated 8 sugar of HA (HA8)
GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAc
Saturated 10 sugar of HA (HA10)
GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAc
Saturated 12 sugar of HA (HA12)
GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAc
The mixture (HA2-18) of saturated 2～18 sugar of HA
The mixture (HA8-14) of saturated 8～14 sugar of HA
HA (weight-average molecular weight 840,000; HA84)
·Gal(6S)β1-4GlcNAc(6S)(L4)
·Gal(6S)β1-4GlcNAc(6S)β1-3Gal(6S)β1-4GlcNAc(6S)(L4L4)
The saturated oligose of HA has used the HA oligose fraction for preparing in aforementioned preparation example 1.
In addition, the unsaturated oligose of HA is by utilizing Chondroitin A C-1 lyase (chondroitinase AC I Huang; Seikagaku Kogyo Co. Ltd. makes) handle HA and degradation production, (with reference to the preparation example 2) that obtains by the method classification the same with aforementioned preparation example 1.
L4 and L4L4 modulate by the method that the open WO96/16973 in the world is put down in writing.
With substances, with according to the test of following pharmacological effect fixed concentration be dissolved in normal saline solution.Be dissolved in the endotoxin concns after the normal saline solution, any one all below 0.3EU/ml, and iron level any one all below 20ppm.
(2) cell, substratum
K562 cell (JCRB0019; People's leukemia cell), PC-12 cell (JCRB0733)
The K562 cell utilizes the RPMI1640 substratum that contains 10% fetal bovine serum (FBS) to cultivate.
In addition, the PC-12 cell, the DMEM substratum that utilization contains 10% horse serum and 5% newborn calf serum (FCS) (contains D-glucose (1,000mg/l), L-glutaminate (4mM), Sodium.alpha.-ketopropionate (110mg/l), sodium bicarbonate (3.7g/l)) among the 575ml, added the substratum of 20% D/W of 10ml and cultivated.
＜1〉HSF1 is to the effect of phosphorylation
Known HSF1 (thermal shock factor 1) protein is the transcription factor (J.Biol.Chem., 271,3355-3358 (1996)) of Hsp70.And HSF1 protein, by being activated by phosphorylation, this phosphorylation detects based on the bands of a spectrum displacement in the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (apparent molecular weight approximately moves to about 81kDa from 66kDa).Utilize this fact, the oligosaccharide act of all size in the K562 of stress application cell, by detecting the displacement of above-mentioned bands of a spectrum, has been determined phosphorylation (activation) degree of HSF1.
(1) experiment 1
With HA4, Δ HA4, HA6, HA8, HA2-18 and HA8-14, be added on separately in the nutrient solution that contains aforementioned cell, make its concentration reach 1,10 or 100ng/ml, apply thermal shocking, incubation 20 minutes by moving to immediately in 42 ℃ the environment afterwards.After the incubation, reclaim cell, carry out SDS-PAGE (gel strength is 10%) by centrifugation.Then use anti-HSF1 monoclonal antibody (manufacturing of Stressgene company) as an antibody, use sheep anti mouse monoclonal antibody IgG (manufacturing of Jackson Lab company) to carry out immunoblotting (Western Blotting), detected bands of a spectrum (HSF1 that does not have phosphorylation) to the displacement of the bands of a spectrum (HSF1 of phosphorylation) of 81kDa from 66kDa as secondary antibodies.
Its result has added in the cell of HA4, Δ HA4, HA6 and HA2-18, has found displacement to the 81kDa bands of a spectrum according to the addition of HA oligose.In HA8-14, do not find obvious variation.Found the obstruction of bands of a spectrum displacements on the contrary for HA8.
(2) experiment 2
In order to confirm to test 1 reproducibility, use HA4, Δ HA4, HA8 and HA84, except the HA addition of nutrient solution only is set at 100ng/ml, thermal shocking is outside carrying out under 43 ℃, and is all the same with experiment 1, detected the bands of a spectrum displacement.Its result is illustrated among Figure 41.In Figure 41, several first swimming lanes are the extract of the cell that (do not add the HA oligose) under 37 ℃ of conditions from a left side, second swimming lane is the extract of the cell that (do not add the HA oligose) under 43 conditions, and the rightmost swimming lane be with the result of HSF1 (from bacterium) with same method detection as standard.
According to Figure 41, in the cell that has added HA4 and Δ HA4, find the bands of a spectrum displacement of HSF 1, and 66 and the two the amount of HSF1 of 81kDa itself also increase.On the other hand, found that in HA8 the amount of the HSF1 of 66kDa increases, but do not found bands of a spectrum displacement to 81kDa.And in HA84, do not find to change.
(3) experiment 3 (comparative experimentss)
In order to determine whether observed effect is special to the sugar chain skeleton structure of HA oligose in experiment 2, carried out using the comparative experiments of following chrondroitin oligose.Chrondroitin (hereinafter referred to as Ch), the GlcNAc that has only HA is different with HA by GalN residue (GalNAc) displacement this point, reach for mucopolysaccharide sulfur-bearing acidic group not aspect identical with HA.Thereby, we can say that Ch is and the extremely similar material of HA structure, for they oligose too.
Saturated 4 sugar of Ch (Ch4)
GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc
Saturated 6 sugar of Ch (Ch6)
GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc
Saturated 8 sugar of Ch (Ch8)
GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAB1-3GalNAc
The saturated oligose of these Ch is according to method (Carbohyd.Res., 141 of Nagasawa etc., the 99-110 page or leaf, 1985), handle the degradation production that Ch obtains, carry out classification with anion-exchange chromatography according to size and obtain by the methyl-sulphoxide (DMSO) that utilization is contained HCl.
Use these oligose, carried out and experiment 2 identical experiments.Its result adds in the cell of any one Ch oligose, does not all observe the bands of a spectrum displacement of HSF1 or the increase of this height of HSF1.
From this experiment as can be seen, the Ch oligose does not have the activation of the HSF1 that finds in the HA oligose or expresses enhancement.This presentation of results, such effect are specific to the sugar chain skeleton structure of HA oligose.
These description of tests, HA4 sugar and contain the fraction (HA2-18) of this material activates HSF1 extremely significantly.
＜2〉effect that Hsp is expressed
Because according to＜1〉determined that HA4 sugar is compared with other big or small HA and can express the significantly activation of the essential factor (HSF1) of Hsp70, so expression for the Hsp in the K562 cell of determining to be applied in stress, with use other big or small HA to compare whether in fact significantly to increase, carried out following experiment by the effect of HA4 sugar.
With HA2, Δ HA4, HA6, HA84 and L4L4, be added on separately in the nutrient solution (37 ℃) that contains aforementioned cell, make its concentration reach 1,10 or 100ng/ml.Apply down thermal shocking, incubation 20 minutes by the environment that moves to 43 ℃ immediately afterwards.Make its nutrient solution return to 37 ℃ of incubations 2 hours again afterwards, reclaim cell by centrifugation afterwards, with above-mentioned＜1 similarly carry out SDS-PAGE.Then use anti-Hsp72 monoclonal antibody (manufacturing of Amersham company) as an antibody, use goat anti-rabbit igg monoclonal antibody (manufacturing of Jackson Lab company), to carry out immunoblotting, detected Hsp72 as secondary antibodies.Hsp72, the most representative for one of Hsp70 family member, find that it is expressed to be induced by stress and strengthen.
The result is illustrated among Figure 42.Leftmost swimming lane is for applying thermal shocking (37 ℃) in a of Figure 42, and do not add substances, and detects the result of Hsp72 equally, and rightmost swimming lane is that same examination criteria Hsp72 is (from the Hsp72 of bacterium; BHsp72) result.The result that b expresses for the Hsp72 that detects equally when having added Δ HA4 in the cell that does not apply thermal shocking in Figure 42.
From result shown in a of Figure 42 as can be seen, when applying thermal shocking (43 ℃) (several second swimming lanes) from a left side, with 37 ℃ under when not adding substances (leftmost swimming lane) compare, the expression of Hsp is enhanced.And the expression of Hsp further is enhanced in the presence of each HA oligose, and the expression of Hsp is enhanced (several the 3rd～5 swimming lanes from a left side) especially forcefully in the presence of Δ HA4.
As mentioned above, added in the cell (having applied the cell of thermal shocking) of Δ HA4 and found the strongly expressed of Hsp, strengthened but added the expression that does not have discovery in the cell of other big or small HA as add the so strong Hsp72 of Δ HA4.
And in the cell that does not apply thermal shocking, there is not to find to strengthen the expression of Hsp72 by Δ HA4.This presentation of results, HA4 sugar is in the almost not influence of expression that does not have under the condition of stress for Hsp, in case stress application is very fast and significantly strengthen the expression of Hsp.
＜3〉restraining effect of necrocytosis
According to above-mentioned experiment, determined that HA4 sugar strengthens the effect that Hsp expresses in the stress cell down and is better than other big or small HA oligose, so, carried out following experiment in fact can definite HA oligose suppress to be subjected to the death of the cell of stress more forcefully.
It is known that the PC-12 cell in the nutrient solution that does not contain serum apoptosis takes place.
Therefore with Δ HA2, HA4, Δ HA4, HA6, Δ HA6, HA8, HA10, HA12, HA84, L4, L4L4, be added on separately and contain in the PC-12 cell nutrient solution of (not containing serum), make its concentration reach 100ng/ml, use trypan blue (trypan blue) with cell dyeing in cell after 24 hours, calculated ratio with respect to total cell count existence cell (the painted cell of trypan blue of no use) number.
Its result is illustrated among Figure 43.Result according to Figure 43 can judge, HA4 sugar, particularly HA4 are compared with other big or small HA oligose or other kind oligose, significantly suppress necrocytosis.
＜4〉cell tissue provide protection
1. the cell of organ preservation aspect, organization protection's effect
(1) modulation of organ preservative fluid
Euro-Collins liquid (the Euro-Collins liquid that will contain 100ng/ml HA4 sugar; Am.J.Surg., 179,154-160 page or leaf (2000)) as testing liquid (below be designated as " HA4 (+) ").In contrast, use Euro-Collins liquid (below be designated as " HA4 (-) ").
(2) administration of substances etc.
With the SD male rat in 11 ages in week, be divided into " HA4 (+) " use group (n=5), " HA4 (-) " use group (n=5), reach normal group (n=5).Open abdomen and exteriorize (liver) afterwards,, carry out perfusion, until whole liver color being shoaled, afterwards 37 ℃ of following incubations 2 hours because of hemorrhage with each solution of about 40ml for " HA4 (+) " use group and " HA4 (-) " use group.
(3) estimate
After the incubation, organ wet weight/the dry weight of calculating each group is than (index of the edema degree that tissue injury causes), with observation by light microscope (visual inspection tissue injury degree) the painted tissue slice of HE, carry out that chromatin concentrates image analysis and according to TUNEL method (the breach end-labelling (Terminal deoxynucleotidyltransferase (TdT)-mediated nick end labeling method) of terminal deoxynucleotidyl transferase mediation; J.Cell.Biol., analysis (evaluation of necrocytosis degree) 119,493-501 page or leaf (1992)).
(3-1) result of the wet weight of organ/dry weight ratio is illustrated among Figure 44.In Figure 44 *There is significant difference in expression because of p＜0.05 (WILLIAMS-DARLING Ton multiple range test (Williams multiple range test)).
According to the result of Figure 44, " HA4 (+) " use group is compared with " HA4 (-) " use group, has significantly reduced wet weight/dry weight ratio.This presentation of results, HA4 sugar can effectively suppress the tissue injury that the preservation by organ causes and the edema that causes.
(3-2) carried out the painted tissue slice of HE with observation by light microscope, the result is, observed edema, histolysis (cytoplasmic disappearance), nuclear and concentrate etc. in " HA4 (-) " use group.In contrast, in " HA4 (+) " use group, observed and normal group image much at one, do not observed observed significant tissue injury in " HA4 (-) " use group.
(3-3) the spissated image analysis of chromatin
As a kind of method of estimating the necrocytosis degree, use and carried out the painted tissue slice of HE, carried out the spissated image analysis of cyto-chromatin (Image-Pro PLUSTM Version3.0.1; Media Cybernetics makes), compared image color.Necrocytosis takes place and chromatin when concentrating, this part is by the HE deep dyed color.The spissated cell of chromatin is many more, and the concentration liquid of MIcrosope image integral body is high more, so it is estimated as index.
Its result is illustrated among Figure 45.In addition in Figure 45, *There is significant difference in expression because of p＜0.05 (WilliamsMultiple range test).
(3-4) the TUNEL method is analyzed
As a kind of method of estimating the necrocytosis degree, carried out utilizing the segment degree analyzing of the DNA of TUNEL method.During cell generation apoptosis, DNA can segmentization be known.The TUNEL method is the terminal painted method with DNA, DNA by the cell of segmentization for a long time, the painted degree of TUNEL can increase, so can be used as the index of necrocytosis (apoptosis) degree.
Tissue slice after preserving is carried out TUNEL dyeing, use the opticmicroscope visual inspection then, counted the TUNEL staining cell number of each square centimeter.Simultaneously, with tissue disorder's degree of tissue slice separately, estimate with " severe ", " moderate ", " slightly ", " extremely slight " 4 stages.And this evaluation, carry out at random in order to ensure objectivity.The former result is illustrated among Figure 46, and the latter's result is illustrated among Figure 47.And in Figure 46 *There is significant difference in expression because of p＜0.05 (Williams multiple range test).
As can be seen, with respect to observe extremely many TUNEL staining cells in " HA4 (-) " use group, the TUNEL staining cell is few in " HA4 (+) " use group, almost is the degree identical with normal group from the result of Figure 46.And as can be seen from Figure 47, in 80% tissue slice, observed the tissue disorder of " severe ", " moderate " in " HA4 (-) " use group, but in " HA4 (+) " use group, do not observe the tissue disorder of " severe ", in 20% tissue slice, observed the tissue disorder of " moderate " at most.
Above presentation of results, HA oligose (HA4 sugar) has good cell, organization protection's effect aspect the organ preservation.
2. in the cell aspect the effect of ulcer, organization protection's effect
(1) modulation of the testing liquid of administrable
Substances is dissolved in the carboxymethyl cellulose (CMC), as the testing liquid of administrable.According to dosage described later, determine concentration, and make to amount of liquid medicine and reach 10ml/kg.
As positive control solution, use in addition existing gastritis, stomach ulcer therapeutical agent (teprenone, geranylgeranylacetone; Trade(brand)name Selbex Eisai) is dissolved in material among the CMC.Determine concentration, make dosage reach 1mg/kg (clinical consumption), and reach 10ml/kg to amount of liquid medicine.Used 0.5%CMC as negative control solution.
(2) administration of substances etc.
SD male rat with 5 ages in week is divided into " negative control solution " administration group (n=8), " HA44ml/kg " administration group (n=8), " HA420mg/kg " administration group (n=8), " HA4100mg/kg " administration group (n=8) and [positive control solution] administration group (n=8).
Make since 16:00 the day before yesterday of the administration first time and respectively to organize mouse and go on a hunger strike, 8:00 limited with water logging after the above-mentioned solution oral administration in second day.The water logging restriction lasts till second day 8:00 (24 hours) always.In the meantime, with administration is the same for the first time each solution is carried out administration at 16:00 and 24:00.
Dissect each mouse afterwards, take out stomach.The stomach that takes out injects 10% buffering formaldehyde solution makes its expansion, and it is sliding and fixing that pleat is flattened.Afterwards, washing is removed attached to the blood on the coat of the stomach.
(3) estimate
Utilize image analysis apparatus (Image-pro PLUSTM Version 3.0.1; The MediaCybernetics manufacturing) measured the area occupation ratio (ulcer area/glandular stomach area) that produces the ulcer portion of chocolate sex change.Its result is illustrated among Figure 48.In Figure 48 *And *Represent separately to have significant difference because of p＜0.05 and p＜0.01 (Williams multiple range test).
As can be seen from Figure 48, HA4 administration group is compared with negative control solution administration group, the area occupation ratio of any one ulcer portion all obviously descends.Particularly HA420mg/kg administration group and HA4100mg/kg administration group, ulcer portion area occupation ratio is also lower than positive control solution group.
Above presentation of results, HA oligose (HA4 sugar) aspect the effect of ulcer, has good cell, organization protection's effect.And the oral administration by the HA oligose, can obtain above-mentioned good effect.And then this limits with water logging after HA oligose (HA4 sugar) administration in advance in experiment, and, need certain hour to producing ulcer after the water logging restriction beginning, so this result of experiment explanation HA oligose (HA4 sugar) has preventive effect.Restriction
3. in the cell aspect the effect of hepatopathy, organization protection's effect
The experiment of tetracol phenixin model is used in experiment 1
(1) modulation of administrable testing liquid
Substances is dissolved in the normal saline solution, as the administrable testing liquid.According to dosage described later, determine concentration, make to amount of liquid medicine to reach 5ml/kg.
And, used FAD (flavin adenine dinucleotide with dosage 10mg/kg as positive control solution; The Wakamoto pharmacy), FAD is for the effect of aftermentioned tetracol phenixin hepatopathy model be in the news (pharmacotherapy, the 9th volume, special collection 46-53 page or leaf).And used normal saline solution as negative control solution.
(2) administration of substances etc.
SD male rat with 5 ages in week is divided into " negative control solution " administration group (n=8), " HA42ml/kg " administration group (n=8), " HA410mg/kg " administration group (n=8), " HA450mg/kg " administration group (n=8) and " positive control solution " administration group (n=8), " normal group " (n=8).
Make since 16:00 the day before yesterday of the administration first time that respectively to organize mouse jejunitas, per os is given with tetracol phenixin (CCl before second day 8:00 4) 100mg/kg.Afterwards at 8:00 with above-mentioned each solution intraperitoneal administration.Still keep the state of going on a hunger strike afterwards, with administration is the same for the first time each solution is carried out administration at 16:00 and 24:00.Carry out feeding after the 24:00 administration, 8:00 dissected each mouse in second day.
(3) estimate
After the dissection, gather blood, measured GOT activity and white blood cell count.The active mensuration of GOT is used clinical chemistry automatic analysing apparatus (COBAS MIRA S; Japan Roche), the mensuration of white blood cell count has been used automatic blood cell determinator (Sysmex (K)-2000; Sysmex company makes).
The former result is illustrated among Figure 49, and the latter's result is illustrated among Figure 50.Among Figure 49 and Figure 50 *There is significant difference in expression because of p＜0.05 (Williams multiple range test).
According to Figure 49 as can be seen, HA450mg/ml administration group is compared with negative control solution administration group, has found the active decline of significant GOT.And according to Figure 50 as can be seen, any one HA4 administration group is compared with negative control solution administration group, has found the minimizing of significant tip white blood cell count, and comparing white blood cell count with FAD also has minimizing.This presentation of results, HA4 sugar has cell, organization protection's effect aspect the effect of hepatopathy.And then, in this experiment tetracol phenixin is given after the usefulness in advance, with HA oligose (HA4 sugar) administration, but with after the tetracol phenixin administration to needing certain hour because of tetracol phenixin produces hepatopathy, this experimental result illustrates that HA oligose (HA4 sugar) has preventive effect.
And in this experiment, IL-10 in the tip blood and the concentration of IL-8 have been measured in the lump.
IL-10; it is reported and (expressed when cell is subjected to stress with stress protein matter; protein with protection cytosis) relevant (J.Immunol. of a kind of Hsp70; 164 (5); 2711-2717 page or leaf (2000)); and there is IL-10 to suppress the report (Autoimmunity, 31 (2), 75-83 page or leaf 1999) of the hepatitis that causes because of concanavalin A.
In addition, in the hepatopathy model that cadmium or ethanol cause, it is known (Toxicology and Applied Pharmacology, 163,231-9 page or leaf (2000) that the expression amount of IL-8 rises, ActaGastro-Enterologica Belgica, 57 (3-4), 255-9 page or leaf (1994)), and report (the Journal of Immunology that suppresses the expression of IL-8 by the expression of inducing Hsp70 is arranged, 164,5416-23 page or leaf (2000)).
IL-10 in the tip blood measures with IL-10Rat ELISA (production of ENDOGEN company).IL-8 measures with Panatest A Series Rat CINC-1 (IL-8) (the Panapharm Laboratories of Co., Ltd.).The measurement result of IL-10 is illustrated among Figure 51, and the measurement result of IL-8 is illustrated among Figure 52.In Figure 51 and Figure 52 *There is significant difference in expression because of p＜0.01 (Williams multiple range test).
As can be seen from Figure 51, the IL-10 concentration in the blood increases with the dosage of HA4, and HA450mg/kg administration group is compared with negative control solution administration group, and IL-10 concentration significantly improves in the blood.And as can be seen from Figure 52, the IL-8 concentration in the blood reduces with the dosage of HA4.
This presentation of results, HA oligose (HA4 sugar) has the effect that promotes the effect that IL-10 produces and suppress the IL-8 generation.Therefore HA oligose (HA4 sugar) produces by promoting the IL-10 generation or suppressing IL-8, can bring into play aforementioned cell, organization protection's effect.And HA oligose (HA4 sugar), for the disease that the increase because of the minimizing of IL-10 or IL-8 causes, maybe need to promote IL-10 to produce or suppress the disease that IL-8 produces etc., can be used as the treatment agent utilization.
The experiment of concanavalin A model is used in experiment 2
Whether effective in order to determine HA oligose (HA4 sugar) to the hepatopathy model except the tetracol phenixin model, carried out using the experiment of concanavalin A model.
(1) modulation of administrable solution
The modulation of administrable testing liquid, concentration, dosage, and to amount of liquid medicine etc., identical with experiment 1.
And, used powerful Neo-Minophangen C (the Stronger Neo-Minophagen C of dosage 5mg/kg as positive control solution; The Minophagen pharmacy).This amount is the highest clinical consumption of this medicament.And used normal saline solution as negative control solution.
(2) administration of substances etc.
Balb/c mouse with 8 ages in week is divided into " negative control solution " administration group (n=12), " HA42ml/kg " administration group (n=12), " HA410mg/kg " administration group (n=12), " HA450mg/kg " administration group (n=12) and " positive control solution " administration group (n=12), " normal group " (n=8).
With concanavalin A (ConA; Sigma company) 15mg/kg is through the tail vein administration.Afterwards with above-mentioned each solution through the tail vein administration.Raised afterwards 24 hours.
(3) estimate
Take blood from abdominal aortic, use clinical chemistry automatic analysing apparatus (COBASMIRA S; Japan Roche) GPT activity and GOT activity have been measured.Take out liver in addition, the state of the liver that detected by an unaided eye.
Its result, GPT mean value is approximately 5600 (I.U./L) in " negative control solution " administration group.Relative therewith, in " HA410mg/kg " administration group and " HA450mg/kg " administration group, be approximately 2200 (I.U./L) and 1200 (I.U./L) separately, any one is all than " negative control solution " administration group (p＜0.01 significantly on the low side; The multiple comparisons of Dunnett detects).In addition, " positive control solution " administration group is approximately 4000 (I.U./L).
The mean value of GOT is approximately 6000 (I.U./L) in " negative control solution " administration group.Relative therewith, in " HA410mg/kg " administration group and " HA450mg/kg " administration group, be approximately 2000 (I.U./L) and 1000 (I.U./L) separately, any one is all than " negative control solution " administration group (p＜0.01 significantly on the low side; The multiple comparisons of Dunnett detects).In addition, " positive control solution " administration group is approximately 4400 (I.U./L).
The visual inspection result of liver is, " HA410mg/kg " administration group and " HA450mg/kg " administration group are compared with " negative control solution " administration group and " positive control solution " administration group, and any one group of obstacle degree all reduces.
These presentation of results, HA oligose (HA4 sugar) is also brought into play effect for the hepatopathy that concanavalin A causes, and this has hinted that HA oligose (HA4 sugar) is effective for various hepatopathies.And then this experiment show the same with the tetracol phenixin model, HA oligose (HA4 sugar) also has preventive effect.
From the high security of HA oligose itself and the result of the foregoing description, can infer the security of various preparations of the present invention.
Industrial applicibility
Oligose of the present invention is useful as the effective constituent of medicine of the present invention.
Fraction of the present invention, for containing the HA oligose of required size, the HA oligose fraction that does not contain other big or small oligose and other impurity in fact as Study on Physiological Activity reagent, analysis standard, medicine itself or its material of HA oligose, is exceedingly useful.
With oligose of the present invention is the medicine of effective constituent, Hsp expression facilitator particularly, and also as can be seen, the HA oligose of specific size is brought into play the unexistent significant effect of other big or small HA oligose from the result of previous embodiment.And then by selecting the HA oligose of this specific size, can in the effect of keeping with other big or small HA oligose, can reduce dosage, aspect cheap and safe more preparation, be exceedingly useful.
And then; with oligose of the present invention is cell death inhibitor, cell obstacle inhibitor, and the cell, organization protection's agent (for example organ preservatives, ulcer treatment agent, hepatopathy treatment agent, IL-10 produce promotor or IL-8 produces inhibitor) of effective constituent; also as described above shown in the result of embodiment; bring into play good pharmacological effect, and safe.

Claims (5)

1. a fraction contains the hyaluronic acid oligosaccharide that the hyaluronic acid tetrose promptly comprises 4 saccharide residues, it is characterized in that, the described physico-chemical property in (1)～(6) below having,
(1) utilize gel filtration chromatography, with following (a) and (b) described detection method analyze this grade timesharing, no matter all show single in fact peak, and as described in the area at this peak reaches (b) as following (a) with any method,
When (a) detecting based on the absorption of 210nm, with respect to whole peak area sum of hyaluronic acid oligosaccharides in the fraction, the relative area at the peak of hyaluronic acid tetrose is more than 85%,
When (b) utilizing differential refractometer to detect, with respect to the peak area sum of whole hyaluronic acid oligosaccharides in the fraction, the relative area at the peak of hyaluronic acid tetrose is more than 98%,
(2) utilize anion-exchange chromatography, analyze this grade timesharing, show single in fact peak based on the absorption detecting of 210nm, and, with respect to the peak area sum of whole hyaluronic acid oligosaccharides in the fraction, the relative area at the peak of hyaluronic acid tetrose is more than 90%
(3) the hyaluronic acid tetrose in this fraction is carried out when analyzing, show single bands of a spectrum after the fluorescent mark by electrophoresis, and, do not detect the bands of a spectrum of other big or small HA oligose,
(4) will constitute the single isotopic molecule amount of hyaluronic acid tetrose of this fraction or the theoretical value of molecular-weight average and be set at 1 o'clock, the measured value of the mass spectroscopy of this fraction counts 0.997～1.003 with relative value,
(5) constitute the carbon of representing by symbol C in the hyaluronic acid tetrose of this fraction, the hydrogen of representing by symbol H and by the theoretical value of the ratio of content separately of the nitrogen of symbolic representation, the measured value of each element that obtains with ultimate analysis poor by this fraction, all in ± 1 weight % scope and
(6) do not contain protein, DNA and intracellular toxin in fact.
2. fraction according to claim 1 is characterized in that, further has as following (7) described physico-chemical property,
(7) this fraction 1H-NMR reaches 13The C-NMR measurement result, with the structure of the hyaluronic acid tetrose shown in the following formula (1) contradiction not,
Formula (1)
Figure C018124320003C1
In the formula, n represents 1, and M represents proton or monovalent cation, and Ac represents ethanoyl.
3. medicine, the fraction of hyaluronic acid tetrose that contains claim 1 be as activeconstituents, and it is to be selected from heat shock protein expression facilitator, cell death inhibitor, cell obstacle inhibitor, and the medicine of cell and organization protection's agent.
4. medicine according to claim 3 is characterized in that, described cell, organization protection's agent are selected from organ preservatives, ulcer treatment agent, hepatopathy treatment agent, IL-10 generation promotor, reach IL-8 generation inhibitor.
5. according to the medicine of claim 3, it is cell and organization protection's agent.
CNB018124321A 2000-07-07 2001-07-06 Hyaluronic acid oligosaccharide fractions and drugs containing the same Expired - Fee Related CN100369925C (en)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
JP206404/2000 2000-07-07
JP206404/00 2000-07-07
JP2000206404 2000-07-07
JP2000247840 2000-08-17
JP247840/2000 2000-08-17
JP247840/00 2000-08-17

Related Child Applications (1)

Application Number Title Priority Date Filing Date
CNA2007103053104A Division CN101229186A (en) 2000-07-07 2001-07-06 Hyaluronic acid oligosaccharide fractions and drugs containing the same

Publications (2)

Publication Number Publication Date
CN1440418A CN1440418A (en) 2003-09-03
CN100369925C true CN100369925C (en) 2008-02-20

Family

ID=26595584

Family Applications (1)

Application Number Title Priority Date Filing Date
CNB018124321A Expired - Fee Related CN100369925C (en) 2000-07-07 2001-07-06 Hyaluronic acid oligosaccharide fractions and drugs containing the same

Country Status (9)

Country Link
US (5) US7507723B2 (en)
EP (1) EP1300412B1 (en)
JP (1) JP4861596B2 (en)
CN (1) CN100369925C (en)
AT (1) ATE453655T1 (en)
AU (3) AU2001269478B2 (en)
CA (2) CA2698561C (en)
DE (1) DE60140941D1 (en)
WO (1) WO2002004471A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109517012A (en) * 2018-11-13 2019-03-26 华熙福瑞达生物医药有限公司 A kind of preparation method of high-purity hyaluronic acid oligosaccharide

Families Citing this family (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7511026B2 (en) 2003-03-25 2009-03-31 Seikagaku Corporation Therapeutic agent for nerve damage
JP4576583B2 (en) * 2005-03-22 2010-11-10 キユーピー株式会社 Hyaluronic acid or a salt thereof, method for producing the same, and cosmetics and food compositions containing the same
JP2007084509A (en) * 2005-09-26 2007-04-05 Toshitsu Kagaku Kenkyusho:Kk Cell activator
US20070099867A1 (en) * 2005-05-24 2007-05-03 Glycoscience Laboratories, Inc. Pharmaceutical agent containing hyaluronan as an active ingredient
JP2007153761A (en) * 2005-12-01 2007-06-21 Toshitsu Kagaku Kenkyusho:Kk Synapse transmission promoter and synapse protective agent
CA2560844C (en) * 2005-09-26 2011-05-17 Glycoscience Laboratories, Inc. Pharmaceutical agent containing hyaluronan as an active ingredient
JP2009091248A (en) * 2006-01-17 2009-04-30 Toshitsu Kagaku Kenkyusho:Kk Therapeutic agent for traumatic neuropathy and/or motor function disorder
KR101482800B1 (en) * 2006-02-24 2015-01-14 큐피가부시키가이샤 Novel low molecular weight hyaluronic acid and/or salt thereof, and cosmetic preparation, pharmaceutical composition, and food composition each using same
JP5457675B2 (en) * 2006-10-17 2014-04-02 株式会社ヒアルロン酸研究所 Therapeutic or preventive for atopic dermatitis
BRPI0621470A2 (en) * 2006-12-05 2011-12-13 Glycoscience Lab Inc therapeutic agent for degenerative arthritis
US20100290989A1 (en) * 2007-10-12 2010-11-18 London Health Sciences Centre Research Inc. Compositions affecting hyaluronic acid mediated activity
CN101326963B (en) * 2008-03-03 2011-06-22 北京神州圣业科技有限公司 Alkaline kation feed immunopotentiator solution, production method and uses thereof
JPWO2009113512A1 (en) * 2008-03-11 2011-07-21 キユーピー株式会社 SOCS3 expression promoter, pharmaceutical and food containing the same, and method for promoting SOCS3 expression
TW201031749A (en) 2009-02-10 2010-09-01 Otsuka Chemical Co Ltd IgG-Fc fragment and method for manufacturing the same
JP5587637B2 (en) * 2009-04-10 2014-09-10 生化学工業株式会社 Matrix metalloprotease inhibitors and uses thereof
CA2871993A1 (en) 2012-02-22 2013-08-29 Hyaluronan Research Institute, Inc. Sirtuin inducer, tissue repairing agent, hepatocyte growth factor inducer, tissue homeostasis maintenance agent, and tlr4 agonist, having hyaluronic acid fragment as active ingredient
EP2698636A1 (en) * 2012-08-13 2014-02-19 Fundació Institut d'Investigació Biomèdica de Bellvitge Methods and reagents for prevention and/or treatment of transplant rejection
WO2015120223A1 (en) * 2014-02-06 2015-08-13 New York University Method for separating hyaluronan and quantifying its molecular mass distribution in biological samples
KR20180028787A (en) * 2016-09-09 2018-03-19 삼성전자주식회사 Defect inspection system and method, and method for fabricating semiconductor using the inspection method
CN110446511B (en) 2017-03-10 2024-04-02 北卡罗来纳大学查珀尔希尔分校 Short acting heparin-based anticoagulant compounds and methods
JP7285048B2 (en) * 2017-03-31 2023-06-01 ロート製薬株式会社 Method for producing composition containing hyaluronic acid oligosaccharide
US11993627B2 (en) 2017-07-03 2024-05-28 The University Of North Carolina At Chapel Hill Enzymatic synthesis of homogeneous chondroitin sulfate oligosaccharides
JP7495061B2 (en) 2017-11-03 2024-06-04 ザ ユニバーシティ オブ ノース カロライナ アット チャペル ヒル Sulfated oligosaccharides with anti-inflammatory properties
US11633424B2 (en) 2018-06-20 2023-04-25 The University Of North Carolina At Chapel Hill Cell protective methods and compositions
CN109528753B (en) * 2018-12-20 2021-01-22 四川大学华西医院 Application of oligomeric hyaluronic acid or salt thereof in preparation of medicine for treating myocardial infarction
CN113876623B (en) * 2021-09-24 2024-06-14 华熙生物科技股份有限公司 Application of hyaluronic acid oligosaccharide composition in resisting skin aging and promoting collagen production
CN115774066A (en) * 2022-11-16 2023-03-10 南京乐韬生物科技有限公司 Method for analyzing component proportion of ultralow molecular weight hyaluronic acid

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09227386A (en) * 1996-02-16 1997-09-02 Seikagaku Kogyo Co Ltd Stress protein expression enhancer
JPH11246301A (en) * 1998-02-27 1999-09-14 Denki Kagaku Kogyo Kk Organ preserving solution
JPH11310588A (en) * 1998-04-30 1999-11-09 Maruha Corp New blood platelet cohesion aggregation inhibitory substance, its production and its use
JP2000103738A (en) * 1998-09-28 2000-04-11 Maruha Corp Promoter for vascular endothelial cell growth

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4141973A (en) * 1975-10-17 1979-02-27 Biotrics, Inc. Ultrapure hyaluronic acid and the use thereof
JPH06107538A (en) * 1992-03-31 1994-04-19 Shiseido Co Ltd Bulb of eye preserving solution for cornea graft
US5902795A (en) * 1992-06-16 1999-05-11 Trustees Of Tufts College Oligosaccharides reactive with hyaluronan-binding protein and their methods of use
US5616568A (en) * 1993-11-30 1997-04-01 The Research Foundation Of State University Of New York Functionalized derivatives of hyaluronic acid
IT1287227B1 (en) * 1996-04-04 1998-08-04 Fidia Spa In Amministrazione S HYALURONIC ACID AS A COMPONENT OF THE CORNEA PRESERVATION LIQUID
CN1185352C (en) * 1998-04-30 2005-01-19 吗鲁哈株式会社 Compounds having glucuronic acid derivatives and glucosamine derivatives in structure, process for producing same and utilization thereof
US7511026B2 (en) * 2003-03-25 2009-03-31 Seikagaku Corporation Therapeutic agent for nerve damage

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09227386A (en) * 1996-02-16 1997-09-02 Seikagaku Kogyo Co Ltd Stress protein expression enhancer
JPH11246301A (en) * 1998-02-27 1999-09-14 Denki Kagaku Kogyo Kk Organ preserving solution
JPH11310588A (en) * 1998-04-30 1999-11-09 Maruha Corp New blood platelet cohesion aggregation inhibitory substance, its production and its use
JP2000103738A (en) * 1998-09-28 2000-04-11 Maruha Corp Promoter for vascular endothelial cell growth

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109517012A (en) * 2018-11-13 2019-03-26 华熙福瑞达生物医药有限公司 A kind of preparation method of high-purity hyaluronic acid oligosaccharide
CN109517012B (en) * 2018-11-13 2020-05-22 华熙生物科技股份有限公司 Preparation method of hyaluronic acid oligosaccharide

Also Published As

Publication number Publication date
JPWO2002004471A1 (en) 2004-01-15
US20080182983A1 (en) 2008-07-31
CA2698561A1 (en) 2002-01-17
EP1300412A1 (en) 2003-04-09
US20110224169A1 (en) 2011-09-15
US20050090661A1 (en) 2005-04-28
AU2001269478B2 (en) 2006-09-21
CA2698561C (en) 2012-07-10
EP1300412B1 (en) 2009-12-30
AU6947801A (en) 2002-01-21
US7507723B2 (en) 2009-03-24
CN1440418A (en) 2003-09-03
US7601488B2 (en) 2009-10-13
CA2414211C (en) 2011-08-02
US20090291914A1 (en) 2009-11-26
DE60140941D1 (en) 2010-02-11
WO2002004471A1 (en) 2002-01-17
AU2006252178B2 (en) 2010-05-27
JP4861596B2 (en) 2012-01-25
EP1300412A4 (en) 2004-08-18
US8314079B2 (en) 2012-11-20
CA2414211A1 (en) 2003-01-06
ATE453655T1 (en) 2010-01-15
AU2006252178A1 (en) 2007-01-18
US20070134646A1 (en) 2007-06-14

Similar Documents

Publication Publication Date Title
CN100369925C (en) Hyaluronic acid oligosaccharide fractions and drugs containing the same
Wieruszeski et al. Structure of the monosialyl oligosaccharides derived from salivary gland mucin glycoproteins of the Chinese swiftlet (genus Collocalia). Characterization of novel types of extended core structure, Gal beta (1—3)[GlcNAc beta (1—6)] GalNAc alpha (1—3) GalNAc (-ol), and of chain termination,[Gal alpha (1—4)] 0-1 [Gal beta (1—4)] 2GlcNAc beta (1—.).
Conrad Methylation of Carbohydrates with Methylsulfinyl Anion and Methyl Iodide in Dimethyl Sulfoxide: Methylation of Aerobacter aerogenes A3 (S1) Capsular Polysaccharide and 3-O-α-d-Glucopyranosyluronic acid-d-mannose
Ribeiro et al. A sulfated α-L-fucan from sea cucumber
Rao et al. Sialyl Lewis X mimics derived from a pharmacophore search are selectin inhibitors with anti-inflammatory activity.
Yu et al. Enzymatic preparation and structural determination of oligosaccharides derived from sea cucumber (Acaudina molpadioides) fucoidan
Dua et al. Characterization of the oligosaccharide alditols from ovarian cyst mucin glycoproteins of blood group A using high pressure liquid chromatography (HPLC) and high field 1H NMR spectroscopy.
Ghosh et al. Sulfated xylomannans from the red seaweed Sebdenia polydactyla: structural features, chemical modification and antiviral activity
ITMI962505A1 (en) NEW BUTYRICS ESTERS WITH ANTI-POLIFERATIVE ACTIVITY AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN THEM
Moreau et al. Application of high-resolution NMR spectroscopy to the elucidation of the structure of the specific capsular polysaccharide of Streptococcus pneumoniae type 7F
Feng et al. Structural characterization and immunological activities of a novel water-soluble polysaccharide from the fruiting bodies of culinary-medicinal winter mushroom, Flammulina velutipes (Agaricomycetes)
EP2985298B1 (en) Low-molecular-weight fucosylated glycosaminoglycan derivative containing terminal 2,5-anhydrated talose or derivative thereof
CN104725520A (en) Diaphragma juglandis acidic polysaccharose and preparation and application thereof
Du Penhoat et al. An NMR solution study of the mega-oligosaccharide, rhamnogalacturonan II
Šimek et al. How the molecular weight affects the in vivo fate of exogenous hyaluronan delivered intravenously: A stable-isotope labelling strategy
Defaye et al. The behavior of cellulose, amylose, and β-D-xylan towards anhydrous hydrogen fluoride
Wang et al. Effect of orally administered hydroxypropyl chitosan on the levels of iron, copper, zinc and calcium in mice
JPH06506973A (en) Sulfated polysaccharides as inhibitors of smooth muscle cell proliferation
ITCO960031A1 (en) LOW MOLECULAR WEIGHT FUCANS WITH ANTICOAGULANT, ANTITROMBINIC AND ANTITROMBOTIC ACTIVITIES
Chiovitti et al. A nearly idealized 6′-O-methylated η-carrageenan from the Australian red alga Claviclonium ovatum (Acrotylaceae, Gigartinales)
CN101229186A (en) Hyaluronic acid oligosaccharide fractions and drugs containing the same
Danishefsky et al. Mucopolysaccharides in animal tumors
CN102603911B (en) Corbicula fluminea glycosaminoglycan and preparation method and usage thereof
Saliternik-Givant et al. Biochemical heterogeneity of the corneal glycosaminoglycans
Laurent Hyaluronan research in Uppsala

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20080220

Termination date: 20150706

EXPY Termination of patent right or utility model